Development of a device and method for the time-course estimation of low water fluxes and mean surface water activity of food products during ripening and storage

Accurate measurement of water activity (a_w) is an important goal for the food industry because a_w is a key parameter in microbial growth, biological reaction rates and physical properties. An experimental device was setup using air-product water balance to non-destructively estimate the time-course of mean a_w at the food product surface under well-controlled airflow conditions. The device is especially suited for studying the ripening of cheeses and fermented meat products, where water fluxes exchanged between products and air are very low. The validation tests performed with a_w-known model products showed that water fluxes of 10^?7 kg s^?1 can be estimated with an accuracy better than 2% over very short periods of time, and that surface a_w can be estimated with an absolute uncertainty of less than 0.01 a_w units. A handful of cheese ripening trials illustrate the potential of the method, highlighting the effects of a low air velocity and high air RH on the water exchanges occurring at a cheese surface, thus demonstrating the strong surface sensitivity to external air conditions.     

Survival of Escherichia coli O157:H7 during Manufacture and Storage of White Brined Cheese

Escherichia coli O157:H7 is a major foodborne pathogen that causes severe disease in humans. Survival of E. coli O157:H7 during processing and storage of white brined cheese was investigated. Cheeses were prepared using pasteurized milk inoculated with a 4 strain E. coli O157:H7 cocktail (7 log₁₀ CFU/g) with or without yogurt starter culture (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus) and stored in 10% or 15% NaCl brine at 10 and 21 ºC for 28 d. NaCl concentration, water activity (a_w), pH, and numbers of E. coli O157:H7 and lactic acid bacteria (LAB) were determined in cheese and brine. E. coli O157:H7 was able to survive in cheese stored in both brines at 10 and 21 ºC regardless of the presence of starter LAB, although the latter significantly enhanced E. coli O157:H7 reduction in cheese or its brine at 10 ºC. E. coli O157:H7 numbers were reduced by 2.6 and 3.4 log₁₀ CFU/g in cheese stored in 10% and 15% NaCl brine, respectively, in the presence of starter LAB and by 1.4 and 2.3 log₁₀ CFU/g, respectively, in the absence of starter LAB at 10 ºC. The pathogen survived, but at lower numbers in the brines. The salt concentration of cheese stored in 10% brine remained about 5% during ripening, but in 15% brine, the NaCl level increased 1.6% to 8.1% (w/w) by 28 d. Values of pH and a_w slightly decreased 1 d after exposure to brine and reached 5.5 to 6.6 and 0.88 to 0.94, respectively, in all treatments.     

The effect of two types of mould inoculants on the microbiological composition,physicochemical properties and protein hydrolysis in two Gorgonzola‐type cheese varieties during ripening

The aim of this paper was to investigate the role of two types of Penicillium roqueforti moulds (type esportazione and dolce) in the ripening of two Gorgonzola‐type cheese varieties. Cheeses were analysed after 4, 14, 30 and 60 days of ripening. Microbiological analysis showed high numbers of total bacterial count, yeasts and moulds in both 60‐day‐old cheese varieties. The concentration of water‐soluble N, nonprotein N and 5% phosphotungstic acid‐soluble N increased significantly during ripening. Patterns of proteolysis by urea‐polyacrylamide gel electrophoresis showed that rind‐to‐core gradients and age‐related changes in moisture and salt content influenced mould and other enzyme activities, which are reflected in various rates of protein degradation. The hydrolysis of αs₁‐ and β‐caseins was more extensive in the core than under the rind of both cheese varieties.     

Determining salt concentrations for equivalent water activity in reduced-sodium cheese by use of a model system

The range of sodium chloride (salt)-to-moisture ratio is critical in producing high-quality cheese products. The salt-to-moisture ratio has numerous effects on cheese quality, including controlling water activity (a_w). Therefore, when attempting to decrease the sodium content of natural cheese it is important to calculate the amount of replacement salts necessary to create the same a_w as the full-sodium target (when using the same cheese making procedure). Most attempts to decrease sodium using replacement salts have used concentrations too low to create the equivalent a_w due to the differences in the molecular weight of the replacers compared with salt. This could be because of the desire to minimize off-flavors inherent in the replacement salts, but it complicates the ability to conclude that the replacement salts are the cause of off-flavors such as bitter. The objective of this study was to develop a model system that could be used to measure a_w directly, without manufacturing cheese, to allow cheese makers to determine the salt and salt replacer concentrations needed to achieve the equivalent a_w for their existing full-sodium control formulas. All-purpose flour, salt, and salt replacers (potassium chloride, modified potassium chloride, magnesium chloride, and calcium chloride) were blended with butter and water at concentrations that approximated the solids, fat, and moisture contents of typical Cheddar cheese. Salt and salt replacers were applied to the model systems at concentrations predicted by Raoult's law. The a_w of the model samples was measured on a water activity meter, and concentrations were adjusted using Raoult's law if they differed from those of the full-sodium model. Based on the results determined using the model system, stirred-curd pilot-scale batches of reduced- and full-sodium Cheddar cheese were manufactured in duplicate. Water activity, pH, and gross composition were measured and evaluated statistically by linear mixed model. The model system method accurately determined the concentrations of salt and salt replacer necessary to achieve the same a_w as the full-sodium control in pilot-scale cheese using different replacement salts.     

Physicochemical differences between Croatian cheese matured in a lamb skin sack (Sir iz misine) and cheese matured in a rind throughout ripening

The aim of this study was to research differences in physicochemical parameters between Croatian cheese in a lamb skin sack (Sir iz misine) and cheese in a rind throughout ripening. Cheese in a sack had significantly (P < 0.05) lower content of total solids, fat, proteins and salt which showed the ‘protective’ effect of skin sack and higher permeability of natural rind. The water‐soluble nitrogen in the total nitrogen (%TN) and 12% trichloroacetic acid‐soluble nitrogen (%TN) at the end of ripening was significantly (P < 0.05) higher in cheese in a sack than in cheese in a rind which indicates intensive proteolysis in cheese in a sack.     

Changes in water binding in high-pressure treated cheese,measured by TGA (thermogravimetrical analysis)

Garrotxa cheese was pressurised at 400 MPa for 5 min to shorten ripening accordingly to previous studies. Moisture content was followed along ripening. Moisture in cheese was divided into free and bound water according to the weight loss rate during drying in a TGA oven. High-pressure treated cheese to shorten ripening time retained more moisture than control cheese. Bound water remained unaffected by the pressure treatment, whereas free water was higher in treated cheese during ripening. Free water content was linked with a_w.     

Model for changes in weight and dry matter during the ripening of a smear soft cheese under controlled temperature and relative humidity

A model predicting the weight loss and the dry matter increase in the core and in the rind of a French smear soft cheese (Munster type) has been developed. Experimental data were collected from experiments carried out in an aseptic pilot scale ripening chamber under nine different combinations of temperature (8, 12 and 16 °C) and of relative humidity (85%, 93% and 99%) according to a complete experimental design. For each run 16 samples were analysed at various ripening stages. The model took into account the water loss by evaporation, the water transfer from the core to the surface and the mass loss due to the CO₂ release. The cheese weight loss was more influenced by the relative humidity than by the temperature. The model was able to account for the experimental weight loss as well as the rind and the core dry matter, with accuracy acceptable for practical use.     

Behaviour of Escherichia coli strains during semi-hard and hard raw milk cheese production

The behaviour of two Escherichia coli strains, one with a high and the other with a low thermotolerance phenotype, was investigated during production and ripening of Swiss semi-hard and hard raw milk cheese. In semi-hard cheese, counts of E. coli increased during production, before a log-linear decrease occurred during ripening, with a faster rate of reduction in core than in rind samples, and faster reduction of the more heat-sensitive strain in rind samples. Nevertheless, at the end of semi-hard cheese ripening, E. coli were present at least at 1.3 log₁₀ cfu g⁻¹ in rind samples and remained detectable by enrichment of core samples. During the first day of hard cheese production, both E. coli strains were almost completely inactivated. Detection by enrichment was possible in one of twelve spiked cheeses after 16 weeks, indicating the potential of a thermotolerant E. coli strain to survive until the end of ripening.     

Antagonistic effects of Lactobacillus reuteri against Escherichia coli O157:H7 in white-brined cheese under different storage conditions

This study aimed to investigate the survival of the foodborne pathogen Escherichia coli O157:H7 in white-brined cheeses as influenced by the presence of Lactobacillus reuteri. The white cheeses were made from pasteurized bovine milk inoculated with E. coli O157:H7 (cocktail of 3 strains) to achieve ～5 log₁₀ cfu/g with absence or presence of Lb. reuteri (～6 log₁₀ cfu/g). Cheese samples were brined in 10% or 15% NaCl solution and stored at 10°C and 25°C for 28 d. The white-brined cheeses were assessed for salt content, pH, water activity (A_w), and numbers of E. coli O157:H7, Lb. reuteri, nonstarter lactic acid bacteria (NSLAB), yeasts, and molds. Results showed that E. coli O157:H7 survived in cheese stored in both brine solutions at 10°C and 25°C regardless of the presence of Lb. reuteri. A substantial reduction was observed in cheese stored in 10% NaCl brine at 25°C, followed by cheese stored in 15% NaCl brine at 10°C by 2.64 and 2.16 log₁₀ cfu/g, respectively, in the presence of Lb. reuteri and by 1.02 and 1.87 log₁₀ cfu/g, respectively, in the absence of Lb. reuteri under the same conditions. The pathogen in brine solutions survived but at a lower rate. Furthermore, the growth of Lb. reuteri and NSLAB were enhanced or slightly decreased in cheese and brine by 28 d, respectively. The salt concentrations of cheese ranged from 4 to 6% and 5 to 7% (wt/wt), during 28-d ripening in 10 and 15% brine, respectively. Values of pH and A_w slightly increased at d 1 after exposure to brine and reached 4.69 to 6.08 and 0.91 to 0.95, respectively, in all treatments. Therefore, the addition of Lb. reuteri can be used as a biopreservation method to inhibit the survival of E. coli O157:H7 in white-brined cheese when combined with the appropriate temperature, NaCl level, and storage time.     

Ectoine as a natural component of food: detection in red smear cheeses

Ectoine is a compatible solute accumulated in halophilic bacteria in response to high salt concentrations and offers protection from osmotic stress. The occurrence of compatible solutes is widespread among bacteria, yet ectoine has never been detected in foods. The use of an ectoine producing microorganism (Brevibacterium linens) in the surface ripening of red smear cheeses led to the question whether ectoine can be found in cheese. Therefore we examined samples from a variety of cheese manufacturers and different types of red smear cheeses for the presence of ectoine using HPLC and HPLC/MS analysis. Ectoine solely appears in the rind and was detected up to 178 mg/200 g red smear cheese, depending on several factors like ripening status and conditions throughout the cheese production process (e.g. salt concentrations of used brine baths).     

Kinetics of Casein Degradation during Ripening of Fynbo Cheese Salted with NaCl/KCl Brine

First-order kinetics with respect to the α_s1-casein concentration was used to study casein degradation during low-fat Fynbo cheese ripening. Effects of partial NaCl replacement by KCI during cheese salting were studied by statistical treatment of the casein degradation results. Four zones from cheeses at 1, 5, 10, 20, and 30 ripening days were analyzed by a polyacrylamide gel electrophoresis method. Similar kinetic parameters were obtained for a cheese salted with a NaCl/KCl brine and for a control cheese during ripening. Results were more affected by salt concentration than by salt substitution. KCl did not strongly influence kinetics of Fynbo cheese proteolysis.     

A model describing Debaryomyces hansenii growth and substrate consumption during a smear soft cheese deacidification and ripening

A mechanistic model for Debaryomyces hansenii growth and substrate consumption, lactose conversion into lactate by lactic acid bacteria, as well as lactose and lactate transfer from the core toward the rind was established. The model described the first step (14 d) of the ripening of a smear soft cheese and included the effects of temperature and relative humidity of the ripening chamber on the kinetic parameters. Experimental data were collected from experiments carried out in an aseptic pilot scale ripening chamber under 9 different combinations of temperature (8, 12, and 16°C) and relative humidity (85, 93, and 99%) according to a complete experimental design. The model considered the cheese as a system with 2 compartments (rind and core) and included 5 state evolution equations and 16 parameters. The model succeeded in predicting D. hansenii growth and lactose and lactate concentrations during the first step of ripening (curd deacidification) in core and rind. The nonlinear data-fitting method allowed the determination of tight confidence intervals for the model parameters. The residual standard error (RSE) between model predictions and experimental data was close to the experimental standard deviation between repeated experiments.     

A Study of Staphylococcus Aureus Growth in Model Systems and Processed Cheese

A survey of a_w and pH in commercial samples of processed cheese produced in Argentina, indicated the a_w varied between 0.950–0.978 and pH ranged from 5.2–6.1 Staphylococcus aureus growth studies in model systems (laboratory media with added NaCl) with a_w and pH adjusted to resemble that of commercial samples of processed cheese showed that S. aureus grows very well at a temperature of 30°C, but complete growth inhibition occurs at refrigerator temperature. The results obtained with model systems were confirmed by S. aureus growth experiments in inoculated cheese.     

On the selection of relevant environmental factors to predict microbial dynamics in solidified media

Several studies have shown that food structure causes slower growth rates and narrower growth boundaries of bacteria compared to laboratory media. In predictive microbiology, both a_w or corresponding solute concentration (mainly NaCl) have been used as a growth influencing factor for kinetic models or growth/no growth interface models. The majority of these models have been based on data generated in liquid broth media with NaCl as the predominant a_w influencing solute. However, in complex food systems, other a_w influencing components might be present, next to NaCl. In this study, the growth rate of Salmonella typhimurium was studied in the growth region and the growth/no growth response was tested in Tryptic Soy Broth at 20 °C at varying gelatin concentration (0, 10, 50 g L⁻¹ gelatin), pH (3.25–5.5) and water activity (a_w) (0.929–0.996). From the viewpoint of water activity, the results suggest that NaCl is the main a_w affecting compound. However, gelatin seemed to have an effect on medium a_w too. Moreover, there is also an interaction effect between NaCl and gelatin. From the microbial viewpoint, the results confirmed that the a_w decreasing effect of gelatin is less harmful to cells than the effect of Na⁺ ions. The unexpected shift of the growth/no growth interface to more severe conditions when going from a liquid medium to a medium with 10 g L⁻¹ gelatin is more pronounced when formulating the models in terms of a_w than in terms of NaCl concentrations. At 50 g L⁻¹ gelatin, the model factored with NaCl concentration shifts to milder conditions (concordant to literature results) while the model with a_w indicates a further shift to more severe conditions, which is due to the water activity lowering effect of gelatin and the interaction between gelatin and NaCl. The results suggest that solute concentration should be used instead of a_w, both for kinetic models in the growth region and for growth/no growth interface models, if the transferability of models to solid foods is to be increased.     

Camembert-type cheese quality and safety implications in relation to the timing of high-pressure processing during aging

Bloomy rind cheeses, including Brie, Camembert, and related varieties, are at high risk of contamination by environmental pathogens during manufacture and ripening. This risk is particularly high during ripening due to open-air exposure of the product. Currently, no kill step is applied after manufacture or post ripening to control food safety risks associated with Listeria monocytogenes contamination. Instead, cheesemakers must rely on sanitation and environmental monitoring to reduce this risk. High-pressure processing (HPP) is a nonthermal food-processing technology that can effectively reduce bacterial contaminants with minimal impact on the organoleptic properties of various foods. The objective of this study was to evaluate HPP as a potential intervention to maintain Camembert cheese quality and reduce risk associated with L. monocytogenes. Timing of HPP treatments (3, 11, and 45 d after manufacture) was based on the growth of L. monocytogenes during Camembert cheese ripening. High-pressure processing treatment of fully ripened cheeses (45 d) resulted in destruction of the surface mold, which caused browning and yellowing of the cheese rind. Applying HPP treatment earlier in the ripening process (11 d) resulted in a similar degradation of cheese appearance, which did not improve with continued ripening. Applying HPP treatment shortly after production (3 d; before the surface flora developed) delayed the development of the cheese rind and the textural ripening of the cheese. This early treatment time also resulted in free whey being expelled from the cheese, creating a firmer body. Applying HPP 11 d after manufacture resulted in >5 log reduction of L. monocytogenes at 450 and 550 MPa with holding times of 10 min. Although HPP was effective at reducing L. monocytogenes associated with bloomy rind cheeses, the quality deterioration would be unacceptable to consumers. Cheesemakers must continue to emphasize sanitation and environmental monitoring to reduce the risk of L. monocytogenes in bloomy rind cheeses.     

Influence of water activity on microbial development in minced,cured pork,in relation to gas headspace composition

The water activity (a_w) of a cured minced meat mixture was reduced with NaCl or glycerol + NaCl and the products stored in air, N₂ or CO₂ at 4°C. The lower the a_w, the lower was the growth rate of the total microflora except when a_w was reduced below 0·98 with glycerol and products were stored in CO₂, when the growth rate was independent of a_w. At a_w 0·94 NaCl was more effective than glycerol + NaCl in reducing the growth rate. In air the final microflora of products stored at a_w ≤0·96 consisted primarily of yeasts while at 0.98 a_w, both Lactobacillus (62%) and yeasts (38%) were found. In CO₂ and N₂Lactobacillus predominated at 0·94–0·98 a_w when 2% NaCl or NaCl + glycerol were used. At 0·94 a_w, produced using 6% NaCl, a heterogenous final microflora was found, including Micrococcus and Staphylococcus. An increase in the microbial shelf-life of meat products may be obtained by reducing the a_w, particularly for use with storage in N₂- and CO₂-atmospheres. The type of solute chosen to regulate a_w may affect the solubility of CO₂, which in turn affects the shelf-life.     

Variation of cardinal growth parameters and growth limits according to phylogenetic affiliation in the Bacillus cereus Group. Consequences for risk assessment

The growth rates of strains covering the seven major phylogenetic groups of Bacillus cereus sensu lato (as defined by Guinebretiere et al., 2008) at a range of temperature (7 °C–55 °C), pH (4.6–7.5) and a_w (0.929–0.996, with 0.5%–10% NaCl as humectant) were determined. Growth rates were fitted by non-linear regression to determine the cardinal parameters T_min, T_opt, T_max, pH_min, pH_opt, a_wmin and μ_opt. We showed that cardinal parameters reflected the differences in the temperature adaptation observed between B. cereus phylogenetic groups I to VII. The ability of growing at low pH (up to 4.3) or low a_w (from a_w 0.929 and up to 10% NaCl) varied among strains. The strains of groups III and VII, the most tolerant to heat, were also the most adapted to high NaCl (all strains growing at 8% NaCl) and the ones of groups I and VI the least adapted (no growth at 7% NaCl). All strains of groups II and VII were able to grow at pH 4.6, and only a few strains of group VI. Phenotypic differences between the two psychrotrophic groups II and VI were revealed by contrasted acid and salt tolerance. The cardinal values determined in this work were validated by comparing with cardinal parameters of a panel of strains published elsewhere and with predictions of growth in a range of foods.     

Controlled production of Camembert-type cheeses. Part I: Microbiological and physicochemical evolutions

A holistic approach of a mould cheese ripening is presented. The objective was to establish relationships between the different microbiological and biochemical changes during cheese ripening. Model cheeses were prepared from pasteurized milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti and Brevibacterium linens under aseptic conditions. Two cheese-making trials with efficient control of environmental parameters were carried out and showed similar ripening characteristics. K. lactis grew rapidly between days 1 and 6 (generation time around 48 h). G. candidum grew exponentially between days 4 and 10 (generation time around 4.6 d). Brevi. linens also grew exponentially but after day 6 when Pen. camemberti mycelium began developing and the pH of the rind was close to 7. Its exponential growth presented 3 phases in relation to carbon and nitrogen substrate availability. Concentrations of Pen. camemberti mycelium were not followed by viable cell count but they were evaluated visually. The viable microorganism concentrations were well correlated with the carbon substrate concentrations in the core and in the rind. The lactose concentrations were negligible after 10 d ripening, and changes in lactate quantities were correlated with fungi flora. The pH of the inner part depended on NH3. Surface pH was significantly related to NH3 concentration and to fungi growth. The acid-soluble nitrogen (ASN) and non-protein nitrogen (NPN) indexes and NH3 concentrations of the rind were low until day 6, and then increased rapidly to follow the fungi concentrations until day 45. The ASN and NPN indexes and NH3 concentrations in the core were lower than in the rind and they showed the same evolution. G. candidum and Pen. camemberti populations have a major effect on proteolysis; nevertheless, K. lactis and Brevi. linens cell lysis also had an impact on proteolysis. Viable cell counts of K. lactis, G. candidum, Pen. camemberti and Brevi. linens were correlated with the environmental conditions, with proteolytic products and with carbon substrate assimilation. NH3 diffusion from surface to the cheese core during ripening was highly suspected. Interaction phenomena between microorganisms are discussed.