Hard surfaces decontamination of enteropathogenic and Shiga toxin-producing Escherichia coli using bacteriophages

Three Myoviridae phages (DT1, DT5 and DT6) specific for pathogenic Escherichia coli were studied, either individually or as cocktails, for their lytic activity on in vitro challenge tests. Also, cocktail ability to reduce artificial contamination on hard surfaces (glass coverslips and stainless steel coupons) by three pathogenic Escherichia coli strains (EPEC920, non-O157 STEC ARG4827 and O157:H7 STEC464) was tested. Assays of phage stability during refrigerated storage showed that the three phages evaluated retained a high viability after two months at 4 °C. Challenge tests showed high reductions in viable cells, of up to 6.4 log CFU ml^− 1, for all tested strains at 37 °C. Efficiency was somewhat lower at 4 °C, though biocontrol levels were still good, reaching values of up to 3.8 log CFU ml^− 1. Considering only results obtained at 37 °C, phage cocktails produced the highest reduction in most cases. Treatments with phage cocktails produced complete inactivation (ca. 5–6 log CFU ml^− 1) of EPEC920 and O157:H7 STEC464 on glass coverslips, and of EPEC920, non-O157 STEC ARG4827 and O157:H7 STEC464 on stainless steel coupons, at both temperatures (4 °C and 37 °C) and multiplicity of infection (ca. 10³ and 10⁷) tested. However, some strains not detected at 3 h were sometimes detected at 24 h, and inactivation of non-O157 STEC ARG4827 on glass coverslips was never accomplished; viable cell reductions in all these cases ranged from 1.2 to 5.4 log CFU ml^− 1. Our results suggest that lytic phages, either individually or as a cocktail, may be useful for reducing contamination on hard materials used in food processing surfaces. To our knowledge, this is the first study focused on the use of bacteriophages to reduce contamination of food processing surfaces by EPEC and non-O157 STEC strains.     

Effect of high hydrostatic pressure on the viability of Streptococcus thermophilus bacteriophages isolated from cheese

The effect of high hydrostatic pressure (HHP) on the inactivation of Streptococcus thermophilus bacteriophages ϕAbc2, ALQ13.2 and DT1 was investigated. Reductions in viability were proportional to pressure (400–600 MPa) and holding time (0–30 min), though the inactivation extent was phage-dependent. HHP treatment at 600 MPa for less than 5 min was sufficient to completely inactivate phages ϕAbc2 and DT1. However, ALQ13.2 only suffered a 3.7-log reduction which showed markedly higher resistance than ϕAbc2 and DT1. Weibull model was applied to describe the inactivation behavior of phages. Regression coefficients (R²), root mean square error (RMSE) and residual plots strongly suggested that Weibull model had a good fit to the data. Additionally, the Weibull models were simplified with a binary model since b values had a linear relationship at 400–600 MPa. The simplified Weibull model provided reasonable predictions of survival data at different pressures for ϕAbc2 and DT1.Industrial relevancePhage infection remains today the most serious and common problem in the manufacture of fermented cheese products. In particular, some S. thermophilus bacteriophages exhibited high thermal resistance, high burst size values and remarkably short latent periods. The use of alternative preservation techniques such as high hydrostatic pressure processing is an efficient method to improve cheese quality and achieve bacteriophages safety. The results showed HHP treatment was capable for complete inactivation of S. thermophilus bacteriophages. The lethal kinetics of S. thermophilus bacteriophages using the Weibull model could help the application of HHP in cheese-making industry.     

Effect of high pressure homogenization on lactic acid bacteria phages and probiotic bacteria phages

To investigate the effect of high pressure homogenization on virus inactivation, phages specific for Lactobacillus delbrueckii, Lactobacillus helveticus, Streptococcus thermophilus, Lactococcus lactis, Lactobacillus paracasei and Lactobacillus plantarum were studied. The influence of pressure, number of passes, suspension medium and phage concentration were studied at 25 °C. Reductions in viability were proportional to pressure and number of passes, though the inactivation extent was phage-dependent. At 100 MPa, some bacteriophages were completely inactivated (6 log₁₀ reduction) after 3 or 5 passes, while others remained infective after 8 passes. For all phages, treatment at 60 MPa was insufficient for complete inactivation, even after 8 passes. No clear influence of suspending medium was observed. Inactivation seems to depend on phage concentration; the higher the initial load, the bigger the reduction achieved. Although these results showed that several phages studied are resistant to high-pressure homogenization, this strategy could be combined with others to control their presence in raw milk.     

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.     

Fate of enterotoxigenic Staphylococcus aureus in the presence of nisin-producing Lactococcus lactis strain during manufacture of Jben,a Moroccan traditional fresh cheese

The inhibitory effect of nisin-producing Lactococcus lactis subsp. lactis UL730 on the growth of enterotoxigenic Staphylococcus aureus J10 during manufacture of Jben, a Moroccan traditional fresh cheese prepared from recombined milk, was investigated.With an inoculum level of 10³ cfu mL⁻¹, S. aureus was absent in Jben four days after inoculation when the nisin-producing lactococcus was used as lactic starter. In contrast, it survived after that period, when the starter was non-nisin-producing. No staphylococcal thermonuclease was detected in all Jben samples made from milk inoculated with S. aureus at the level of 10³ cfu mL⁻¹.With a higher inoculum of 10⁵ cfu mL⁻¹, S. aureus was still present in Jben after manufacture and persisted during the storage of the product for 3 days in the laboratory, even when the starter used was nisin-producing. Staphylococcal thermonuclease and type C enterotoxin were detected in all Jben samples made from milk inoculated with 10⁵ cfu mL⁻¹. Thermonuclease and enterotoxin were already produced in the coagulum, at 24 h after milk inoculation with S. aureus.     

Prevalence of bacteriophages infecting Staphylococcus aureus in dairy samples and their potential as biocontrol agents

The prevalence of bacteriophages infecting Staphylococcus aureus in dairy samples was assessed. Fourteen Staph. aureus strains were used in enrichment cultures of 75 dairy samples. All samples grew specific Staph. aureus bacteriophages. According to the host range, 8 different phages were isolated. Three of them, phages ΦH5, ΦG7, and ΦA72, were found in 89% of the samples; all the isolated phages were temperate. Phages ΦH5 and ΦA72 were used in preliminary bacterial challenge tests against Staph. aureus in milk. A phage mixture (1:1) was more effective than each single phage, most likely by preventing the survival of lysogenized cells. Phages inhibited Staph. aureus in UHT and pasteurized whole-fat milk. However, the phages were less active in semi-skimmed raw milk and little inhibition was achieved in whole, raw milk. Killing of Staph. aureus was observed at room temperature and at 37°C, but not at refrigeration temperature.     

Antibiotic resistance and susceptibility to some food preservative measures of spoilage and pathogenic micro-organisms from spices

Antibiogram of 84 strains of Bacillus cereus, 26 strains of Clostridium perfringens, four strains of Staphylococcus aureus, 51 strains of Enterobacteriaceae, two strains of each of Salmonella and Shigella; isolated from spices, were studied against 20 different antibiotics that are commonly used against foodborne diseases, mainly gastroenteritis. All the tested strains of B. cereus, Cl. perfringens, Staph. aureus, Escherichia coli, Salmonella and Shigella were found resistant to at least 3, 4, 7, 6, 10 and 9 antibiotics, respectively. In brain–heart infusion broth supplemented with glucose, the D_100°C-values for B. cereus were 3.5–5.9 min, and the z-values were 17–18°C. The D_100°C-values for Cl. perfringens in fluid thioglycolate medium were higher (10.0–19.8 min) than those of B. cereus. The minimum inhibitory concentrations (MICs) of sodium chloride were 45–80 mg ml⁻¹. While the MIC of benzoic acid for Cl. perfringens, tested on perfringens agar (pH 7.3) plates by incubating anaerobially at 35°C for 24 h, was 1.9–2.2 mg ml⁻¹, for others, tested on nutrient agar (pH 6.8) plates by incubating at 35°C for 18 h in static aerobic condition, it was much less. Similarly, the MIC of sorbic acid for all the tested isolates, excepting Cl. perfringens, was 0.6–1.1 mg ml⁻¹. Of the eight isolates of Cl. perfringens, only three were inhibited at 2.0 mg sorbic acid ml⁻¹, while others were resistant. Sixty percent and 75% of the respective strains of B. cereus and Cl. perfringens were resistant to 5000 IU Nisaplin ml⁻¹, whereas the MIC values of Staph. aureus were between 3000 and 5000 IU ml⁻¹. While studying combined effect of selected hurdles on the growth of enterotoxigenic Cl. perfringens 16-C2, the judicious combination considered was low acid (pH 6.0), 30 mg sodium chloride ml⁻¹ and 1.25 mg benzoic acid ml⁻¹.     

Radio-sensitivity of pathogens in inoculated prepared foods of animal origin

The effectiveness of irradiation for inactivating Staphylococcus aureus, Listeria ivanovii, Salmonella Typhimurium, and Escherichia coli in the prepared foods of animal origin was investigated. Commercially available seasoned and cooked beef, fried egg, and ham were purchased, radiation-sterilized, and inoculated at 10⁶–10⁷ CFU/g with each of the four pathogens and stored at three storage conditions at 10 °C, 20 °C, and 30 °C. D₁₀-values of S. aureus, L. ivanovii, Salmonella Typhimurium, and E. coli were 0.34±0.01, 0.24±0.02, 0.24±0.01, and 0.27±0.01 kGy, respectively. No viable cells were detected at 3 kGy of irradiation. Salmonella mutagenicity assay (Ames test) indicated that the 10 kGy irradiated samples were statistically similar to non-irradiated control samples in the Salmonella mutagenicity assay (Ames test). These studies demonstrate that irradiation can be used as an additional safety tool to produce microbiologically safe and wholesome prepared foods of animal origin.     

Evaluation of a cocktail of three bacteriophages for the biocontrol of Salmonella of wastewater

Salmonella serovars are increasing in importance as significant pathogens of both human and animals. Although water and wastewater are treated to eliminate pathogenic microorganisms, they still play an important role in the transmission of Salmonella spp. In this study, bacteriophages infecting Salmonella spp. were isolated from wastewater and evaluated; for their potential to lyse environmental Salmonella strains in vitro at different MOIs and temperatures; and to control the wastewater bacterial community. Three distinct phages designated sww65, sww275, and sww297; as defined by plaque morphology, electron microscopy and host range; were obtained from wastewater. Challenge tests were performed at 37, and 30 °C with the infection of the Salmonella cultures with individual phage, a mixture of two phages, and cocktail of three phages at MOIs of 10⁰, 10², and 10⁴ PFU/CFU. At 30, and 37 °C, a cocktail of three phages reduced all of the Salmonella cultures tested. These results required a high multiplicity of infection. However, when infected with only one phage or a mixture of two phages at MOIs of 10⁰ or 10 ² PFU/CFU, an emergence of bacterial resistance was observed. The dynamic monitoring of wastewater enterobacterial community was conducted using Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). The number of bands decreased gradually with the use of individual phage or phage cocktails. Moreover, the dynamic monitoring of Salmonella community during wastewater treatment was performed using PCR detection of virulence gene invA. The results correlated with the ERIC-PCR fingerprints, and suggested that Salmonella community was affected by the phage treatment. Indeed, in wastewater, bacteriophages are reducing Salmonella and other members of the Enterobacteriaceae. These results indicated that dynamic changes are closely related with the process of treatment. The introduction of wide host range bacteriophages in wastewater can have a potential impact on the dynamics of the microbial communities, manifested by the reduction or the elimination of microbial species.     

Phage adsorption on Enteropathogenic and Shiga Toxin-Producing Escherichia coli strains: Influence of physicochemical and physiological factors

Bacteriophages have proved to be useful tools against pathogenic Escherichia coli strains. The first step in the phage life cycle is the adsorption to the host cell surface. In the present work, three Myoviridae phages (DT1, DT5 and DT6) were used to characterize the adsorption process on three pathogenic E. coli strains, namely two Shiga-toxin producing E. coli (STEC) and one enteropathogenic E. coli (EPEC), in several conditions found on food. The influence of Na⁺, Mg^2 +, temperature, pH, periodate, proteinase K and physiological cell state on phage adsorption was investigated. The three phages evaluated showed high adsorption rates at pH 7.5 and 5.7 while they were moderate at the lowest pH evaluated (4.5). Sodium or magnesium ions were not indispensable for the adsorption of the three phages evaluated. Specifically, phage particles were adsorbed either in the presence or absence of Mg^2 +, while increasing Na⁺ concentration resulted in lower adsorption values for all the systems evaluated. Regarding temperature, phage adsorption was slightly affected at 4 °C and 50 °C, while it reached its maximum at 37 °C. Adsorption rates decreased after the thermal inactivation of cells, though, when chloramphenicol (as protein-synthesis inhibitor) was used, adsorption values on treated and untreated cells were similar. In addition, periodate was able to decrease phage adsorption, thus suggesting the receptors were carbohydrates in nature. All these results showed that the adsorption process was only partially affected and most conditions are suitable for the completion of the first step in the phage life cycle. Therefore, phages evaluated in this study can be used to prevent foodborne diseases on several food matrices since they are active in a wide range of environmental conditions.     

Effect of cook temperature on starter and non-starter lactic acid bacteria viability,cheese composition and ripening indices of a semi-hard cheese manufactured using thermophilic cultures

Semi-hard cheeses were manufactured using Streptococcus thermophilus and Lactobacillus helveticus cultures and their ripening was characterised. During cheese manufacture, curds were cooked to a maximum temperature of 47, 50 or 53 °C, pre-pressed under whey at pH 6.15, moulded, pressed and brined. Increased cook temperature resulted in increased manufacture time, a significantly reduced growth rate of S. thermophilus during manufacture in the order 47≈50 °C>53 °C and in significantly lower mean viable cell counts of S. thermophilus up to 56 d of ripening. Increasing cook temperature had no significant effect on mean viable cell numbers of L. helveticus or non-starter lactic acid bacteria (NSLAB). Cheeses produced from curds cooked to 47 °C had significantly higher levels of moisture in non-fat substances (MNFSs), salt-in-moisture and a significantly lower pH and levels of butyrate compared with cheeses produced from curds cooked to 50 or 53 °C.     

Microbial shelf-life of high-pressure-homogenised milk

The anti-bacterial effect of high pressure homogenisation (HPH) on milk is widely reported but the shelf-life of HPH-treated milk, as reported in this communication has not been studied thus far. Raw whole milk was homogenised at 200 or 250 MPa at 55 or 70 °C and counts of total bacteria (TBC), psychrotrophs, pseudomonads, coliforms, lactobacilli, Bacillus cereus and Staphylococcus aureus were determined throughout subsequent storage for 14 days at 4 °C. Immediately after HPH treatment, counts of all bacteria were below the level of detection but after storage for 14 days at 4 °C, TBC, psychrotroph and pseudomonad counts had reached ∼10⁸ cfu mL⁻¹ in all samples treated with HPH. The limited shelf-life obtained indicates that HPH of milk at these processing parameters it is not a suitable alternative to pasteurisation for extending the shelf-life of milk.     

Effect of pre-incubation conditions on growth and survival of Staphylococcus aureus in sliced cooked chicken breast

In this work, the effect of pre-incubation conditions (temperature: 10, 15, 37 °C; pH 5.5, 6.5 and water activity, a_w: 0.997, 0.960) was evaluated on the subsequent growth, survival and enterotoxin production (SE) of Staphylococcus aureus in cooked chicken breast incubated at 10 and 20 °C. Results showed the ability of S. aureus to survive at 10 °C when pre-incubated at low a_w (0.960) what could constitute a food risk if osmotic stressed cells of S. aureus which form biofilms survive on dried surfaces, and they are transferred to cooked meat products by cross-contamination. Regarding growth at 20 °C, cells pre-incubated at pH 5.5 and a_w 0.960 had a longer lag phase and a slower maximum growth rate. On the contrary, it was highlighted that pre-incubation at optimal conditions (37 °C/pH 6.5/a_w 0.997) produced a better adaptation and a faster growth in meat products what would lead to a higher SE production. These findings can support the adoption of management strategies and preventive measures in food industries leading to avoid growth and SE production in meat products.     

Contribution of coagulant and native microflora to the volatile-free fatty acid profile of an artisanal cheese

The contributions of the coagulant Cynara cardunculus and of the microflora of raw milk to the volatile-free fatty acid profile of Serra da Estrela cheese were evaluated. The experimental design included both a model system and, dual cheeses. The study in the model system showed that isovaleric acid was the predominant volatile compound after 7 d of ripening. The systems inoculated with Enterococcus faecium produced the highest amount of this volatile (ca. 135.8 mg kg⁻¹ curd), while those inoculated with Lactobacillus plantarum produced the least (21.4 mg kg⁻¹ curd); Lactococcus lactis produced moderate amounts (ca. 34.2 mg kg⁻¹ curd) but a total amount of volatile-free fatty acids similar to those found in control samples. This is considered advantageous since this volatile fatty acid confers a harsh, piquant, mature flavour to cheese, coupled with the realisation that excess volatiles may result in off-flavours. The addition of cultures in experimental cheeses helped reduce ripening time to about one half. Inclusion of Lb. plantarum led to cheeses containing the highest amounts of volatiles, and exhibiting an aroma closest to that of typical Serra da Estrela cheese.     

Inactivation of bacteriophages by thermal and high-pressure treatment

Dairy companies commonly experience fermentation failures due to bacteriophages that are spread mainly by milk, whey or air. Heat or high-pressure treatment may potentially reduce the phage titre, but further knowledge about the inactivation kinetics is desirable. Inactivation experiments were carried out with the commonly occurring lactococcal phages P001 and P008. Phage suspensions in calcium-enriched M17-broth were heated at 55–80 °C, or high-pressure treated at up to 600 MPa. Kinetic analysis showed that the order of inactivation reaction was above 1; thus, inactivation kinetics were approximated by a non-linear regression model. The Arrhenius parameters, rate constant, k_p,T, and activation energy, E_A (for heat treatments), and the volume of activation, ΔV^# (for pressure treatments) were calculated. Both measured and calculated results indicate that phage P008 was the more heat- and pressure-resistant of the two. By combining the results from heat and pressure inactivations, a pressure–temperature diagram for phage P008 was established.     

Water vapor transport properties during staling of bread crumb and crust as affected by heating rate

In order to develop a mathematical model to simulate mass transfer occurring between the crumb and the crust during bread staling, water vapor sorption properties, i.e., moisture diffusivity, WVP and sorption of bread crumb and crust were investigated at 15 °C. Two types of bread baked with two heating rates (7.39 °C/min and 6.32 °C/min) were considered. Sorption and desorption isotherms were determined using Dynamic Vapor Sorption (DVS) and FF and GAB models were applied in the range of 0–0.95 a_w, to fit isotherm curves. Diffusivity was determined from sorption isotherms by using Fick's law and WVP was measured by two methods (gravimetric and from sorption data). Results exhibited maximum values of D_eff in the range of 0.1 and 0.14 g/g d.b. moisture contents. They varied between 0.88 × 10^? 10 and 0.92 × 10^? 10 m²/s for the crust and between 2.24 × 10^? 10 and 2.64 × 10^? 10 m²/s for the crumb, baked respectively at 220 °C and 240 °C. Results of WVP showed that the crust baked at 240 °C was significantly more permeable than the crust baked at 220 °C. This fact was attributed to the difference in porosity and the molecular structure due to heating effects. Also, the presence of steam in the oven atmosphere enhanced the development of higher porosity in the crust, leading to different structures and properties. Moreover, SEM images showed that starch granules were intact and less swelled in the upper crust when baking at 240 °C, resulting in higher WVP.     

Antimicrobial activity of pediocin-producing Lactococcus lactis on Listeria monocytogenes,Staphylococcus aureus and Escherichia coli O157:H7 in cheese

The antimicrobial activity of two pediocin-producing transformants obtained from wild strains of Lactococcus lactis on the survival of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 during cheese ripening was investigated. Cheeses were manufactured from milk inoculated with the three pathogens, each at approximately 6 log cfu mL⁻¹. Pediococcus acidilactici 347 (Ped⁺), Lc. lactis ESI 153, Lc. lactis ESI 515 (Nis⁺) and their respective pediocin-producing transformants Lc. lactis CL1 (Ped⁺) and Lc. lactis CL2 (Nis⁺, Ped⁺) were added at 1% as adjuncts to the starter culture. After 30 d, L. monocytogenes, S. aureus and E. coli O157:H7 counts were 5.30, 5.16 and 4.14 log cfu g⁻¹ in control cheese made without adjunct culture. On day 30, pediocin-producing derivatives Lc. lactis CL1 and Lc. lactis CL2 lowered L. monocytogenes counts by 2.97 and 1.64 log units, S. aureus by 0.98 and 0.40 log units, and E. coli O157:H7 by 0.84 and 1.69 log units with respect to control cheese. All cheeses made with nisin-producing LAB exhibited bacteriocin activity throughout ripening. Pediocin activity was only detected throughout the whole ripening period in cheese with Lc. lactis CL1. Because of the antimicrobial activity of pediocin PA-1, its production in situ by strains of LAB growing efficiently in milk would extend the application of this bacteriocin in cheese manufacture.     

Screening for and characterization of Lactococcus lactis bacteriophages with high thermal resistance

Fifty-six Lactococcus lactis phage isolates collected from different German dairies and obtained from a starter culture manufacturer were tested for their heat resistance. About 40% of these isolates resisted treatment at 80 °C for 5 min when they were heated in milk. The most resistant phage isolate, P1532, was collected from sour cream. Plaque-formation was still detectable even after heating at 97 °C for 5 min. The second heat-resistant one, P680, showed some plaque-forming ability after heating at 95 °C for 5 min. Kinetic parameters for the thermal inactivation of these two resistant phages were determined for temperatures ranging from 70 to 97 °C. The inactivation of phage P1532 in skim milk and in buffer medium were found to follow first-order kinetics and did not exhibit tailing, whereas in the inactivation curves of phage P680 tailing was observed. The D-value of P1532 at pasteurization temperature of 72 °C was calculated as 112 min.     

Protocol for the manufacture of miniature washed-curd cheeses under controlled microbiological conditions

A protocol for the preparation of miniature washed-curd cheeses under controlled bacteriological conditions was designed and tested for reproducibility. The process was adapted from “Saint-Paulin” technology, and involves inoculation and renneting in autoclaved bottles, and cutting, stirring, curd washing and removal of whey by centrifugation. Pressing was simulated by low-speed centrifugation. All operations were performed using sterile techniques and autoclaved equipment. Forty miniature cheeses (approximately 40 g) were produced over 10 working days, and ripened for 28 days. Gross composition (dry matter, salt-in-moisture and pH) of the one-day-old cheeses did not differ significantly between cheesemaking days, and average values were 45.16 , 2.46 and 5.15%, respectively. Adventitious Lactobacillus population remained less than 200 CFU g⁻¹ all during ripening, and phages were absent. Nitrogen soluble at pH 4.4 and in phosphotungstic acid attained 21 and 3% of total nitrogen, respectively, in 28-day-old cheeses. The proposed model was shown to be suitable for the preparation of miniature cheese specimens for use in microbiological studies of cheese manufacture and ripening.     

Modeling the inactivation kinetics of fruit bromelain in pineapple during high-pressure and thermal treatments

The stability of fruit bromelain (FBM) in pineapple pulp was studied within a high-pressure domain of 0.1–600 MPa/30–70 °C/1 s–30 min. The pulse effect was quantified as a function of pressure, temperature, pressure build-up and decompression times. A maximum of 60% reduction in FBM activity was obtained after a single pulse of 600 MPa/70 °C. Upon applying nth order model, the obtained reaction order (n) for thermal (0.1 MPa/30–70 °C) and high-pressure (100–600 MPa/30–70 °C) inactivation was 1.1 and 1.2, respectively. The inactivation rate constant (k) ranged from 1.2 to 45.0 × 10^− 3 U^n − 1 min^− 1. The activation energy was nonlinearly dependent on pressure (P); whereas, the activation volume was linearly related to temperature (T). The nonlinear dependence of k on P and T was modeled by an empirical equation. The D-values obtained from the empirical model appeared to be more realistic than those from the log-linear kinetics.Industrial relevancePineapple fruit bromelain (FBM) has numerous health benefits and therapeutic effects. It is a protease enzyme that helps in digestion. Processing of pineapple pulp needs attention towards retaining the maximum FBM activity in it. A detailed kinetic study of FBM within a broad range of pressure–temperature–time domain will help in designing a high-pressure process for the pineapple pulp with respect to its bromelain stability.