Applicability of bacteriocin‐producing Lactobacillus plantarum,Enterococcus faecium and Lactococcus lactis ssp. lactis as adjunct starter in Minas Frescal cheesemaking

The antimicrobial and technological characteristics of three bacteriocinogenic cultures used as adjunct starters in Minas Frescal cheese were investigated. The cheeses were manufactured with 1% commercial lactic starter and 0.5%Lactococcus lactis ssp. lactis ATCC 11454, Lactobacillus plantarum ALC 01 or Enterococcus faecium FAIR‐E 198. The cheeses were then artificially inoculated with Listeria monocytogenes Scott A, Staphylococcus aureus ATCC 27154 and Bacillus cereus K1‐B041 and stored for 21 days at 8°C. The results show that there was no significant difference in the counts of L. monocytogenes and S. aureus between the cheeses made with added bacteriocinogenic cultures and the control cheese. On the other hand, B. cereus exhibited susceptibility to Lb. plantarum ALC 01 and E. faecium FAIR‐E 198 from the seventh day of storage. However, these cultures increased the proteolysis of the Minas Frescal cheese.     

Effects of salts of organic acids on the survival of Listeria monocytogenes in soft cheeses

The purpose of this study was to determine the effect of sodium lactate and sodium propionate, both in combination with sodium acetate, on strains of Listeria monocytogenes in artificially inoculated soft cheeses. Minas Frescal and Coalho cheeses, inoculated with a mix of L. monocytogenes 1/2a and Scott A, underwent two treatments: 2% (w/v) sodium lactate in combination with 0.25% (w/v) sodium acetate and 2% (w/v) sodium propionate in combination with 0.25% (w/v) sodium acetate. The samples were analysed immediately and after 7 days at 10 °C. The growth of the pathogen was inhibited in cheeses containing the salts of organic acids, and the effects of treatment were statistically significant (P < 0.05). However, there was no difference between the types of treatment applied. Our data demonstrate that the effectiveness of the salts of organic acids depended on the initial concentration of L. monocytogenes and that a higher concentration of the salts is necessary to ensure sustained inactivation of target pathogens because they are weakly antilisterial when the soft cheeses are stored at 10 °C.     

Survival of Listeria monocytogenes in Galotyri,a traditional Greek soft acid-curd cheese,stored aerobically at 4°C and 12°C

Galotyri is a traditional Greek soft acid-curd cheese, which is made from ewes’ or goats’ milk and is consumed fresh. Because cheese processing may allow Listeria monocytogenes post-process contamination, this study evaluated survival of the pathogen in fresh cheese during storage. Portions (0.5 kg) of two commercial types (<2% salt) of Galotyri, one artisan (pH 4.0±0.1) and the other industrial (pH 3.8±0.1), were inoculated with ca. 3 or 7 log cfu g⁻¹ of a five-strain cocktail of L. monocytogenes and stored aerobically at 4°C and 12°C. After 3 days, average declines of pathogen's populations (PALCAM agar) were 1.3–1.6 and 3.7–4.6 log cfu g⁻¹ in cheese samples for the low and high inocula, respectively. These declines were independent (P>0.05) of the cheese type or the storage temperature. From day 3, however, declines shifted to small or minimal to result in 1.4–1.8 log cfu g⁻¹ of survivors at 28 days of storage of all cheeses at 4°C, indicating a strong “tailing” independent of initial level of contamination. Low (1.2–1.7 log cfu g⁻¹) survival of L. monocytogenes also occurred in cheeses at 12°C for 14 days, which were prone to surface yeast spoilage. When ca. 3 log cfu g⁻¹ of L. monocytogenes were inoculated in laboratory scale prepared Galotyri of pH ≅4.4 and ≅3% salt, the pathogen died off at 14 and 21 days at 12°C and 4°C, respectively, in artisan type cheeses fermented with the natural starter. In contrast, the pathogen survived for 28 days in cheeses fermented with the industrial starter. These results indicate that L. monocytogenes cannot grow but may survive during retail storage of Galotyri despite its low pH of or slightly below 4.0. Although contamination of Galotyri with L. monocytogenes may be expected low (<100 cfu g⁻¹) in practice, that long-term survival of the pathogen in commercial cheeses was shown to be unaffected by the artificial contamination level (3 or 7 logs) and the storage temperature (4°C or 12°C), which should be a concern.     

Application of bacteriocinogenic Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch in the control of Listeria monocytogenes in fresh Minas cheese

Several strains of Enterococcus spp. are capable of producing bacteriocins with antimicrobial activity against important bacterial pathogens in dairy products. In this study, the bacteriocins produced by two Enterococcus strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch), isolated from cheeses, were characterized and tested for their capability to control growth of Listeria monocytogenes 426 in experimentally contaminated fresh Minas cheese during refrigerated storage. Both strains were active against a variety of pathogenic and non-pathogenic microorganisms and bacteriocin absorption to various L. monocytogenes, Enterococcus faecalis ATCC 19443 and Lactobacillus sakei ATCC 15521 varied according to the strain and the testing conditions (pH, temperature, presence of salts and surfactants). Growth of L. monocytogenes 426 was inhibited in cheeses containing E. mundtii CRL35 up to 12 days at 8 °C, evidencing a bacteriostatic effect. E. faecium ST88Ch was less effective, as the bacteriostatic affect occurred only after 6 days at 8 °C. In cheeses containing nisin (12.5 mg/kg), less than one log reduction was observed. This research underlines the potential application of E. mundtii CRL35 in the control of L. monocytogenes in Minas cheese.     

Biofilm formation on stainless steel as a function of time and temperature and control through sanitizers

Enterococcus spp. contamination was screened from a Minas Frescal cheese processing line. Biofilm formation of Enterococcus faecium and Enterococcus faecalis isolates was evaluated and the effect of sanitization procedures in the control of these biofilms was investigated. Enterococcus spp. were detected in raw milk, milk machine, door handle, floor, drain, thermometer, and Minas Frescal cheese. Biofilm formation on stainless steel was modelled as a function of time (0, 1.2, 4, 6.8, and 8 days) and temperature (7, 13, 27, 41, and 47 °C) using response surface methodology. The model showed that E. faecium biofilms were formed from 1 to 8 days at 12–47 °C, while E. faecalis biofilms were formed from 1 to 8 days at 10–43 °C. None of the sanitizers (sodium hypochlorite 100 mg L⁻¹, peracetic acid 300 mg L⁻¹, and chlorhexidine digluconate 400 mg L⁻¹) was able to completely eliminate the biofilms.     

Nisin treatments to control Listeria monocytogenes post-processing contamination on Anthotyros, a traditional Greek whey cheese, stored at 4°C in vacuum packages

Post-processing contamination and growth of Listeria monocytogenes in whey cheeses stored under refrigeration is an important safety concern. This study evaluated commercially available nisin (Nisaplin^®) as a biopreservative to control L. monocytogenes introduced post-processing on Anthotyros, a traditional Greek whey cheese, stored at 4°C in vacuum packages for up to 45 days. The whey used (pH 6.5–6.7) was from Feta cheese manufacture, and it was subjected either to natural acidification (pH 5.3, readjusted to 6.2 with 10% NaOH) prior to heating, or to direct acidification (pH 6.0–6.2) at 80°C with 10% citric acid. Nisin was added either to the whey (100 or 500 IU g⁻¹) prior to heating, or to the cheese (500 IU g⁻¹) prior to packaging, also inoculated with ca. 10⁴ cfu g⁻¹ of L. monocytogenes strain Scott A. In cheese samples without nisin, L. monocytogenes (PALCAM agar) exceeded 7 log cfu g⁻¹ after the first 10 days of storage, irrespective of the whey acidification method. All nisin treatments had an immediate lethal effect (0.7–2.2 log reduction) on L. monocytogenes populations at inoculation (day 0), which was more pronounced with 500 IU g⁻¹ added to the whey. This treatment also suppressed L. monocytogenes growth below the inoculation level for 30 and 45 days in naturally and directly acidified samples, respectively. All other treatments had weak antilisterial effects. Nisin reversed the natural spoilage flora of Anthotyros cheese from Gram-positive to Gram-negative, and this ecological alteration was far more pronounced in the most effective antilisterial treatments.     

Survey of aflatoxin M1 in cheese from the North-east region of São Paulo,Brazil

In the present study, 24 samples of Minas Frescal cheese and 24 samples of Minas Padrão cheese produced in the North-east region of the state of São Paulo, Brazil, were analysed for aflatoxin M₁ (AFM₁) by high-performance liquid chromatography (HPLC) between March and August 2008. AFM₁ was detected in 13 (27.1%) samples at concentrations ranging from 0.037 to 0.313?ng?g^?1. The mean concentrations of AFM₁ in positive samples of Minas Frescal and Minas Padrão cheese were 0.142?±?0.118 and 0.118?±?0.054?ng?g^?1, respectively. It is concluded that the incidence of AFM₁ in Minas cheese may contribute to an increase in the overall ingestion of aflatoxins in the diet, hence indicating the need for the adoption of a tolerance limit for AFM₁ in cheese in Brazil.     

Enhanced antimicrobial activity of nisin in combination with allyl isothiocyanate against Listeria monocytogenes,Staphylococcus aureus,Salmonella Typhimurium and Shigella boydii

This study was designed to evaluate the synergistic antimicrobial effect of nisin and allyl isothiocyanate (AITC) against Listeria monocytogenes, Staphylococcus aureus, Salmonella Typhimurium and Shigella boydii. The synergistic interactions between nisin and AITC were observed against all foodborne pathogens, showing the fractional inhibitory concentrations <1. The populations of L. monocytogenes and S. aureus at the combined treatment of nisin and AITC were decreased to below 1 log CFU mL^?1 after 10‐h incubation at 37 °C. The changes in fatty acid profiles of all strains were substantially influenced by nisin alone and the combined treatment of nisin and AITC. A good agreement was observed among cell viability, membrane permeability and depolarisation activity in response to nisin and AITC. The results suggest that nisin and AITC as synergistic inhibitors could be an effective approach to achieve satisfactory antimicrobial activity against a wide range of foodborne pathogens.     

Effect of probiotic Minas Frescal cheese on the volatile compound and metabolic profiles assessed by nuclear magnetic resonance spectroscopy and chemometric tools

This study aimed to evaluate the effect of Lacticaseibacillus casei 01 as a probiotic culture on the production of volatile organic compounds and metabolic profile of Minas Frescal cheese. Lactose (α-lactose and β-lactose), fatty acids (unsaturated and saturated), citric acid, tryptophan, and benzoic acid were the main compounds. Compared with the control cheese, probiotic cheese was characterized by the highest concentration of tryptophan and presented a higher number of volatile acids. The control cheese was characterized by the highest concentration of benzoic acid and fatty acids, resulting in a higher number of volatile alcohols and esters. No differences were observed for α-lactose, β-lactose, and citric acid contents. A clear separation of probiotic and control Minas Frescal cheese was obtained using ¹H nuclear magnetic resonance spectra, demonstrating that the addition of probiotic culture altered the metabolic profile of Minas Frescal cheese. Overall, the findings suggested that the addition of probiotic culture promoted the proteolysis in the fresh cheeses, decreased the lipolysis, and altered the volatile compounds. Furthermore, nuclear magnetic resonance spectroscopy coupled to chemometrics tools could be used to differentiate probiotic and conventional cheeses.     

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.     

Influence of probiotic strains added to cottage cheese on generation of potentially antioxidant peptides,anti-listerial activity,and survival of probiotic microorganisms in simulated gastrointestinal conditions

Our objective was to evaluate the viability of probiotic microorganisms added to cottage cheese under simulated gastrointestinal conditions, the release of potentially-antioxidant peptides, and their antimicrobial effect on Listeria monocytogenes. Cottage cheeses were prepared in triplicate, incorporating Lactobacillus casei, Lactobacillus rhamnosus GG, the commercial mix YO-MIX™ 205, or a control without probiotic addition. The probiotic population remained at >10⁶ cfu g⁻¹ during 28 days of storage at 8 °C. Cheeses made with added probiotics showed an increased metabolic activity with higher levels of lactic and acetic acids. Higher numbers of potentially bioactive peptides were observed in cheeses added with probiotics. L. monocytogenes population was reduced by about one log cycle after 20 days of storage, in cheeses with probiotics added. Our results indicate that cottage cheese is a good vehicle for probiotic bacteria.     

Application of enterocins A and B, sakacin K and nisin to extend the safe shelf-life of pressurized ready-to-eat meat products

The efficiency of food preservation systems is determined by the technologies that are combined, the intrinsic properties of the food products and the target microorganisms. In the present study, the bacteriocins nisin, enterocins A and B and sakacin K were applied to cooked and dry cured ham spiked with Listeria monocytogenes, Salmonella enterica and Staphylococcus aureus and submitted to a high pressure treatment of 600 MPa. Before pressurization nisin produced significant reductions to the counts of L. monocytogenes and S. aureus, especially in dry cured ham. After the pressurization, Salmonella and L. monocytogenes were not detected in 25 g of both cooked and dry cured ham and remained at this level during the entire storage (57 days at 4 °C + 63 days at 15 °C). S. aureus levels, in contrast, only decreased below the detection limit (1 log CFU/g) in the nisin batches. Afterward, when storage was performed at an abusive temperature, the ability of S. aureus to grow was dependant on the bacteriocin applied and the kind of meat product. Thus, at the end of storage, while S. aureus counts were <1 log CFU/g in all dry cured ham batches, only nisin could inhibit its growth in cooked ham.     

Effect of liposome-encapsulated nisin and bacteriocin-like substance P34 on Listeria monocytogenes growth in Minas frescal cheese

The efficacy of liposome-encapsulated nisin and bacteriocin-like substance (BLS) P34 to control growth of Listeria monocytogenes in Minas frescal cheese was investigated. Nisin and BLS P34 were encapsulated in partially purified soybean phosphatidylcholine (PC-1) and PC-1-cholesterol (7:3) liposomes. PC-1 nanovesicles were previously characterized. PC-1-cholesterol encapsulated nisin and BLS P34 presented, respectively, 218 nm and 158 nm diameters, zeta potential of -64 mV and -53 mV, and entrapment efficiency of 88.9% and 100%. All treatments reduced the population of L. monocytogenes compared to the control during 21 days of storage of Minas frescal cheese at 7°C. However, nisin and BLS P34 encapsulated in PC-1-cholesterol liposomes were less efficient in controlling L. monocytogenes growth in comparison with free and PC-1 liposome-encapsulated bacteriocins. The highest inhibitory effect was observed for nisin and BLS P34 encapsulated in PC-1 liposomes after 10 days of storage of the product. The encapsulation of bacteriocins in liposomes of partially purified soybean phosphatidylcholine may be a promising technology for the control of foodborne pathogens in cheeses.     

Synergistic antimicrobial effect of lactocin AL705 and nisin combined with organic acid salts against Listeria innocua 7 in broth and a hard cheese

The effectiveness of antimicrobial mixtures against Listeria innocua 7, used as a L. monocytogenes surrogate, was investigated in broth and a food system. Synergistic effects were found for nisin (Nis), potassium sorbate (PS), calcium propionate (CP) and sodium lactate (SL), Nis + PS being the most effective binary mixture that exhibited listericidal activity in broth. To assess the effect of adding lactocin AL705 (AL705) to Nis + organic acid salt combinations, tridimensional isobolograms were generated. Sub-MIC combinations of the antimicrobials exerted bactericidal activity against L. innocua 7 after AL705 addition to the binary mixtures. However, when applied on Sardo cheese contaminated with L. innocua 7 (initial inoculum 4.45 ± 0.06 CFU g⁻¹), only Nis + PS + AL705 produced count reductions respect to the control, reaching 3.04 ± 0.35 CFU g⁻¹ counts after 15 days at 15 °C. Ternary combinations containing AL705 showed potential to reduce antimicrobial usages for L. innocua 7 inhibition.     

Effects of treatment with nisin on the microbial flora and sensory properties of a Greek soft acid-curd cheese stored aerobically at 4 °C

The present study evaluated the use of nisin as an antimicrobial treatment for shelf-life extension of Galotyri, a Greek soft acid-curd cheese, stored aerobically under refrigeration for a period of 42 days. Three different treatments were tested: N0, control sample with no nisin added; N1, 50 IU g⁻¹ nisin; and N2, 150 IU g⁻¹ nisin, the latter two treatments added post-production to the Galotyri cheese. Of all microorganisms enumerated, lactobacilli, lactococci and yeasts were the groups that prevailed in cheese samples, irrespective of antimicrobial treatment. Based primarily on sensory evaluation (appearance and taste) and a microbiological acceptability limit for yeasts (5 log cfu g⁻¹), the use of nisin treatments extended the shelf-life of fresh Galotyri cheese stored at 4 °C by ca. 7 days (N1) and 21 days (N2) with cheese maintaining good sensory characteristics.     

Effect of ripening temperature on the growth and significance of non-starter lactic acid bacteria in Cheddar cheese made from raw or pasteurised milk

Two cheese-making trials were conducted, each involving four cheeses, two made from raw milk (R1, R8) and two from pasteurised milk (P1, P8), and ripened at 1°C (R1, P1) or 8°C (R8, P8). The 1-day-old R1 and R8 cheese in trials 1 and 2 contained ∼10⁴ non-starter lactic acid bacteria (NSLAB) g⁻¹. In trial 1, no NSLAB were detected in 1-day-old P1 and P8 cheeses while those in trial 2 contained 10² cfu g⁻¹. In both trials, the maximum differences between the number of NSLAB in the cheeses ripened at 1 or 8°C were observed at 4 months, when the number of NSLAB in cheeses ripened at 8°C were 3 log cycles higher than in those ripened at 1°C. At the end of ripening (6-months), the number of NSLAB in P8 and R8 were ∼2 log cycles higher than in P1 and R1 cheeses, respectively. Primary proteolysis in the cheeses was markedly affected by ripening temperature, but not by pasteurisation of the cheese milk. Urea-polyacyrlamide gel electrophoretograms and reverse-phase (RP)-HPLC of the water-soluble fraction showed differences between cheeses made from raw or pasteurised milk and between cheeses ripened at 1 or 8°C. The concentration of amino acids and fatty acids were in the order R8>P8>R1>P1. Commercial graders awarded highest flavour scores to the R1 cheeses during gradings at 4, 5 and 6 months. A sensory panel found that most flavour and aroma attributes and maturity were in the order of R8>P8>R1=P1. The results of this study suggest that NSLAB play an important role in the development of flavour in Cheddar cheese by contributing to the production of amino acids and fatty acids.     

The occurrence,growth, and biocontrol of Listeria monocytogenes in fresh and surface-ripened soft and semisoft cheeses

Listeria monocytogenes continues to pose a food safety risk in ready-to-eat foods, including fresh and soft/semisoft cheeses. Despite L. monocytogenes being detected regularly along the cheese production continuum, variations in cheese style and intrinsic/extrinsic factors throughout the production process (e.g., pH, water activity, and temperature) affect the potential for L. monocytogenes survival and growth. As novel preservation strategies against the growth of L. monocytogenes in susceptible cheeses, researchers have investigated the use of various biocontrol strategies, including bacteriocins and bacteriocin-producing cultures, bacteriophages, and competition with native microbiota. Bacteriocins produced by lactic acid bacteria (LAB) are of particular interest to the dairy industry since they are often effective against Gram-positive organisms such as L. monocytogenes, and because many LAB are granted Generally Regarded as Safe (GRAS) status by global food safety authorities. Similarly, bacteriophages are also considered a safe form of biocontrol since they have high specificity for their target bacterium. Both bacteriocins and bacteriophages have shown success in reducing L. monocytogenes populations in cheeses in the short term, but regrowth of surviving cells can commonly occur in the finished cheeses. Competition with native microbiota, not mediated by bacteriocin production, has also shown potential to inhibit the growth of L. monocytogenes in cheeses, but the mechanisms are still unclear. Here, we have reviewed the current knowledge on the growth of L. monocytogenes in fresh and surface-ripened soft and semisoft cheeses, as well as the various methods used for biocontrol of this common foodborne pathogen.     

Behaviour of Listeria monocytogenes and Staphylococcus aureus in sliced,vacuum-packaged raw milk cheese stored at two different temperatures and time periods

The behaviour of Listeria monocytogenes and Staphylococcus aureus in raw milk cheese slices packaged under vacuum was evaluated. Artificially contaminated 80-day ripened cheese was portioned, vacuum packaged, and then stored for 28 days at 4 °C and for 56 days at 10 °C. Bacterial counts were obtained before vacuum packaging and then weekly during storage. At the end of ripening, the initial L. monocytogenes count was 4.46 ± 0.89 log cfu g⁻¹; weekly bacterial counts remained substantially unchanged in the samples stored at 4 °C but decreased to 3.54 ± 1.54 log cfu g⁻¹ in those stored at 10 °C. The initial S. aureus count before vacuum packaging was 3.60 ± 0.78 log cfu g⁻¹; it then gradually decreased to 2.60 ± 1.32 log cfu g⁻¹ in the samples stored at 4 °C and to about 1.9 log cfu g⁻¹ in those stored at 10 °C.     

Bactericidal synergism through bacteriocins and high pressure in a meat model system during storage

High hydrostatic pressure represents an attractive non-thermal process for meat products to avoid post-processing contamination. When combined with antimicrobials, like bacteriocins, the death rate may be increased because of sub-lethal injuries to living cells. The behaviour of several foodborne bacteria inoculated in a meat model system with added bacteriocins (enterocins A and B, sakacin K, pediocin AcH or nisin) after pressurization (400 MPa, 10 min, 17°C) and during chilled storage was investigated. Although Staphylococcus was the genus least sensitive to pressurization, the samples including nisin displayed lower and significantly different counts during the 4°C storage than the rest of the treatments. A greater inactivation of Escherichia coli (>6 log₁₀) in the presence of nisin was recorded, the number of survivors remained unchanged during storage at 4°C for 61 days. Nisin was also the bacteriocin capable of maintaining slime-producing lactic acid bacteria below the detection limit (<10² cfu g⁻¹ ). Listeria monocytogenes in treatments with sakacin, enterocins or pediocin was kept <10² cfu g⁻¹ till the end of storage (61 days). Salmonella enterica subsp. enterica ser London and Salmonella enterica subsp.enterica ser Schwarzengrund counts in every treatment were kept at the level obtained after pressurization, with no significant differences between treatments during the chilled storage.