Evaluation of pulsed electric field and minimal heat treatments for inactivation of pseudomonads and enhancement of milk shelf-life

Pulsed Electric Field (PEF) treatment of milk provides the opportunity to increase the shelf-life of fresh milk for distribution to distant markets. PEF treatments were evaluated in sterile (UHT) milk to determine the inactivation of added spoilage Pseudomonas isolates and the subsequent gains in microbial shelf-life (time taken to reach 10⁷ CFU mL^− 1). Little inactivation of Pseudomonas was achieved at 15 or 40 °C compared with 50 or 55 °C. The greatest inactivation (> 5 logs) was achieved by processing at 55 °C with 31 kV cm^− 1 (139.4 kJ L^− 1). Heat treatment at the application temperature without PEF treatment caused minimal inactivation of Pseudomonas (only 0.2 logs), demonstrating that the inactivation of the Pseudomonas was due to the PEF treatment rather than the heat applied to the milk. At added Pseudomonas levels of 10³ and 10⁵ CFU mL^− 1, the microbial shelf-life of PEF-treated milk was extended by at least 8 days at 4 °C compared with untreated milk. The total microbial shelf-life of the PEF-treated milk was 13 and 11 days for inoculation levels of 10³ and 10⁵ CFU mL^− 1 respectively. The results indicate that PEF treatment is useful for the reduction of pseudomonads, the major spoilage bacteria of milk.Industrial relevancePseudomonads are the major psychrotrophic spoilage microflora of refrigerated, stored HTST pasteurised milk. Long-life (UHT) products are an important component of milk sales in South-East Asia, but in recent years there has been an increasing demand for less processed milk products with extended shelf-life. The recent practice of shipping fresh bulk milk from Australia to South-East Asian countries has necessitated additional heat treatment prior to export and on arrival, to achieve the required shelf-life. Pulsed electric field treatment of HTST milk, applied alone or in combination with mild heat under optimised conditions, offers the opportunity of shelf-life extension, while limiting the reduction in quality attributes of milk associated with more severe additional heat treatments.     

The growth and survival of Escherichia coli O157:H7 on minced bison and pieces of bison meat stored at 5 and 10 °C

This study investigated the growth and survival of Escherichia coli O157:H7 on minced and whole pieces of bison meat. Growth curves of native microflora, including Pseudomonas spp. and Enterobacteriaceae were also generated. A marked E. coli O157:H7 strain was inoculated onto minced and whole pieces of bison meat at an initial level of 1.5 log₁₀ cfu g⁻¹. The inoculated meat was stored at either 5 °C for 28 days or 10 °C for 21 days. Survival, but no growth, of E. coli O157:H7 was observed on both forms of bison meat stored at 5 °C, while significant growth of the organism was observed at 10 °C. E. coli O157:H7 counts on whole pieces were generally higher than counts observed on minced bison meat, and reached their highest population by 14 days, with a total increase of 3.36 log₁₀ cfu g⁻¹ on whole pieces and 2.12 log₁₀ cfu g⁻¹on minced bison meat stored at 10 °C. Under the same storage temperature, Pseudomonas spp. and total counts displayed similar growth patterns on both pieces and minced bison meat, while the Enterobacteriaceae showed a slower growth rate. This study showed that the growth of E. coli O157:H7 on bison meat is similar to that observed in studies of beef.     

Effect of thermosonication,pulsed electric field and their combination on inactivation of Listeria innocua in milk

The impact of thermosonication (TS) and pulsed electric field (PEF), individually and combined, on the survival of Listeria innocua 11288 (NCTC) in milk was investigated. TS (400 W, 160 s) without pre-heating reduced L. innocua by 1.2 log₁₀ cfu mL^?1, while shorter treatment times produced negligible inactivation, suggesting TS to be a hurdle rather than an effective standalone treatment. PEF (30 and 40 kV cm^?1, 50 μs) at 10 °C caused a reduction of L. innocua of 1.1 and 3.3 log cycles, respectively. The highest field strength (40 kV cm^?1) combined with TS (80 s) led to 6.8 log₁₀ cfu mL^?1 inactivation. Milk pre-heated to 55 °C (over 60 s) prior to TS followed by PEF (30 and 40 kV cm^?1) showed inactivation between 4.5 and 6.9 log₁₀ cfu mL^?1, the latter being comparable (P > 0.05) with thermal pasteurisation. The data indicate that TS followed by PEF represents a valid alternative for L. innocua inactivation in milk.     

High-pressure and temperature effects on the inactivation of Bacillus amyloliquefaciens,alkaline phosphatase and storage stability of conjugated linoleic acid in milk

Milk rich in conjugated linoleic acid (CLA, 42 ± 3 mg g^− 1 fat) was used to evaluate the impact of high-pressure sterilization (HPS). The pressure, temperature and time needed to reduce 7-log of Bacillus amyloliquefaciens endospores were determined in the presence of nisin (4–64 mg L^− 1). In addition, the inactivation of alkaline phosphatase was evaluated. After HPS treatment, the remaining CLA and formation of hydroperoxides were monitored during storage up to 60 d at 25 °C. The addition of nisin (≥ 16 mg L^− 1) to milk significantly enhanced the inactivation of B. amyloliquefaciens (7-log reduction) after treatment at 600 MPa, 120 °C and 5 min of holding time. These conditions were selected to evaluate the impact of HPS on the CLA retention and hydroperoxides formation. Milk with the addition of nisin and treated with HPS delivered higher retention of CLA and a lower concentration of hydroperoxides compared with the UHT equivalent process (125 °C/15 s and 135 °C/10 s).Industrial relevanceHigh-pressure sterilization is a valuable alternative to produce superior quality milk products in cases where traditional thermal treatments have failed. This study evaluated the impact of processing conditions on the conjugated linoleic acid content at conditions where commercial sterilization has been achieved (7-log reduction of B. amyloliquefaciens). The outcomes of this study are considered as a step further for the development of high-pressure sterilized milk.     

A preliminary study on the role of alkaline phosphatase in cheese ripening

A preparation of exogenous alkaline phosphatase (ALP), containing 17,500 mU L⁻¹, was added to pasteurized milk (PM) to study its role in cheese ripening. Three miniature Cheddar-type cheeses were made from PM containing no added ALP (control), PM plus 23 μL ALP (T1), to give ALP concentration similar to that in raw milk, and PM plus 46 μL ALP (T2). Milk, after addition of ALP, was held at 6 °C for 12 h before cheese manufacture and the experiment was replicated three times. The control, T1 and T2 milks contained ALP activity of 415, 2391 and 4705 mU L⁻¹, respectively. The addition of ALP to PM caused significant (P<0.05) changes in moisture content of miniature cheeses but did not cause any changes in protein content. Levels of water-soluble N during ripening of the cheeses were similar for control, T1 and T2 cheeses. The concentration of amino acids was not affected by the level of ALP present in milk. However, reversed-phase HPLC showed differences in the peptide patterns of control, T1 and T2 cheeses, suggesting a role of ALP in cheese ripening. The results suggest that ALP may play a role in cheese ripening, but further studies are needed to confirm this.     

Effects of high pressure treatment on indigenous enzymes in bovine milk: Reaction kinetics,inactivation and potential application

High pressure-induced inactivation of the indigenous milk enzymes alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and phosphohexoseisomerase (PHI) was studied in the pressure range 400–800 MPa at temperatures between 5 and 40 °C. With respect to pressure stability the following ranking was observed: ALP>GGT>PHI. PHI was inactivated after pressure treatment at 500 MPa and 20 °C for 10 min. In terms of reaction kinetics, inactivation of GGT followed first-order reaction kinetics in the range of 400–800 MPa whereas a reaction order of 1.5 was found for ALP. Reactivation of pressure-treated ALP was observed at low enzyme activity resulting from severe pressure treatment and 2 h storage at 35 °C. The influence of process temperature on the pressure-induced inactivation of GGT and ALP was limited in the range 5–40 °C.     

Numerical and experimental studies on a novel Steinmetz treatment chamber for inactivation of Escherichia coli by radio frequency electric fields

Radio frequency electric field (RFEF) is an emerging non-thermal food processing technology that is alternative to pulsed electric fields (PEF). A novel Steinmetz treatment chamber design, that increases homogeneity of the electric field distribution, has been proposed and constructed. The design was performed and evaluated via Multiphysics modelling. The model was validated by comparison with experimental measurements of outlet temperature, power consumption and current. Inactivation studies showed the influence of electric field intensity, temperature, and treatment time on the inactivation of Escherichia coli in saline water with electrical conductivities between 0.02 and 0.1 S m^− 1. A maximum inactivation of 3.6 log CFU mL^− 1 was obtained at 26.5 kV cm^− 1, 45 °C and 900 μs treatment time. An energy demand of 63 kJ kg^− 1 is required for 1 log reduction, when processing at 25 °C, for foods having 0.1 S m^− 1 conductivity at 20 °C, but energy consumption increases with the electrical conductivity.     

Comparison of the effect of pulsed electric field or high voltage electrical discharge for the control of sweet white must fermentation process with the conventional addition of sulfur dioxide

The present work discusses the efficiency of pulsed electrical treatments for the inactivation of yeasts. The application of pulsed electric fields (PEFs) and high voltage electrical discharges (HVEDs) as alternatives to sulfites, which are used as anti-microbial to stop the fermentation of sweet white wine, was investigated. The influence of sulfite concentration (from 0 mg·L^− 1 to 500 mg·L^− 1), PEF (from 4 kV·cm^− 1 to 20 kV·cm^− 1; from 0.25 ms to 6 ms) and HVED (40 kV/cm; 1 ms or 4 ms) treatments on the inactivation of total yeasts and non-Saccharomyces yeasts was determined. The addition of SO₂ (250 mg·L^− 1) resulted in 8 log total yeast reduction. The maximum yeast inactivation obtained with PEF and HVED was respectively 3 and 4 logs. The use of SO₂, HVED and PEF allows decreasing the non-Saccharomyces yeast level by 7, 5 and 4 logs respectively. However, the wine browning was less pronounced for the samples treated by PEF in comparison with HVED and SO₂ treatments. PEF seems to be the most suitable alternative technique to sulfite addition.     

The inactivation of Listeria monocytogenes by pulsed electric field (PEF) treatment in a static chamber

An experimental analysis of the effect of pulsed electric field (PEF) energy on the inactivation of Listeria monocytogenes was conducted using a custom-designed static chamber and a gel suspension medium for treatment. This allowed PEF energy to be delivered to the suspension under near isothermal conditions. The effects of variations in the number of pulses (5–50 pulses), electric field strength (15–30 kV/cm), temperature (0–60°C) and media bases (water and skim milk) on the inactivation of L. monocytogenes were examined. At temperatures less than 50°C a maximum of 1 log reduction was obtained for L. monocytogenes regardless of pulse number or electric field strength within the ranges examined. In skim milk no reduction occurred. At 50°C and 55°C synergy between PEF and thermal energy was observed. The experimental approach separated the contribution of PEF and thermal energy to total kill and thus allowed this synergy to be quantified. At 55°C the kill due to PEF energy increased to 4.5 logs with another 4.5 logs reduction attributable to thermal energy. It appears that under the conditions of this study PEF alone has a very limited effect on the reduction of L. monocytogenes. However, the addition of thermal energy not only contributed to the kill, but also increased the susceptibility of L. monocytogenes to PEF energy.     

Enrichment of the conjugated linoleic acid content of bovine milk fat by dry fractionation

This study investigated the effect of fat fractionation on the conjugated linoleic acid (cis-9, trans-11-C_18 : 2) content of bovine milk fat. Anhydrous milk fat was fractionated into hard and soft fractions using controlled cooling and agitation. Fractionation of milk fat pre-melted at 60°C using a temperature programme of 33–10°C and a cooling rate of 0.58°C h⁻¹ yielded a soft fraction containing 63.2% more conjugated linoleic acid (2.22 g 100 g⁻¹ FAME), which was also enriched in polyunsaturated fatty acids and vaccenic acid (trans-11-C_18 : 1) compared with the parent fat. Agitation following fractionation was found to have a negative effect on the conjugated linoleic acid content of the soft fraction. Refractionation of the soft fraction did not increase the yield of conjugated linoleic acid. The conjugated linoleic acid and trans fatty acid content of 26 selected food products ranging in milk fat content from 0 to 100% is reported. Conjugated linoleic acid concentrations ranged from 0 to 16.2 mg g⁻¹ fat and were generally lower than the trans fatty acid content which ranged from 0 to 155.7 mg g⁻¹ fat. Spreads containing vegetable oils contained higher trans fatty acid and lower conjugated linoleic acid contents than milk fat-containing products. This study highlights that a milk fat fraction enriched in conjugated linoleic acid may be achieved by dry fractionation.     

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.     

HPLC of proteose peptones for evaluating ageing of packaged pasteurized milk

The proteolysis of casein (CN) occurring in packaged pasteurized milk (PM) during refrigerated storage was studied with relation to hygienic and microbiological characteristics of starting raw milk. Six batches of raw milk having standard plate count (SPC) from 1.5×10⁴ to 2.5×10⁵ cfu mL⁻¹ and somatic cell count (SCC) from 1.6×10⁵ to 4.4×10⁵ units mL⁻¹ were pasteurized (73 °C for 15 s), packaged and stored at 4 °C for 12 days. Capillary zone electrophoresis of CN showed breakdown of β-CN in all PM samples during storage. An HPLC method for monitoring proteose peptones (PP) formation was developed. Level of PP in PM samples increased, with keeping time from 667–789 to 947–1383 mg L⁻¹ and PP formation was significantly (P<0.05) related to SCC of starting raw milk. Electrospray ionization–mass spectrometry showed that PP were mainly represented by PP-5 from either A¹ or A² variants of β-CN. Five commercial samples of PM were analysed for PP formation during 14-day storage at 4 °C. Commercial samples prepared by microfiltration process or bactofugation combined with pasteurization showed the slowest formation of PP. The effect of storage temperature on PP formation was evaluated by keeping a conventional PM sample at either 8 or 12 °C for 12 days. Proteolysis of all major CNs upon action of plasmin and bacterial proteinases was observed under these conditions. PP level thus proves to be a reliable analytical index for evaluating the ageing of packaged PM during refrigerated storage.     

High-pressure homogenisation of raw bovine milk. Effects on fat globule size distribution and microbial inactivation

Whole raw milk was processed using a 15 L h⁻¹ homogeniser with a high-pressure (HP) valve immediately followed by a cooling heat exchanger. The influence of homogenisation pressure (100–300 MPa) and milk inlet temperature T_in (4°C, 14°C or 24°C) on milk temperature T₂ at the HP valve outlet, on fat globule size distribution and on the reduction of the endogenous flora were investigated. The T_in values of 4–24°C led to milk temperatures of 14–33°C before the HP valve, mainly because of compression heating. High T_in and/or homogenisation pressure decreased the fat globule size. At 200 MPa, the d_4.3 diameter of fat globules decreased from 3.8±0.2 (control milk) to 0.80±0.08 μm, 0.65±0.10 or 0.37±0.07 μm at T_in=4, 14°C or 24°C, respectively. A second homogenisation pass at 200 MPa (T_in=4°C, 14°C or 24°C) further decreased d_4.3 diameters to about 0.2 μm and narrowed the size distribution. At all T_in tested, an homogenisation pressure of 300 MPa induced clusters of fat globules, easily dissociated with SDS, and probably formed by sharing protein constituents adsorbed at the fat globule surface. The total endogenous flora of raw milk was reduced by more than 1 log cycle, provided homogenisation pressure was ⩾200 MPa at T_in=24°C (T₂∼60°C), 250 MPa at T_in=14°C (T₂∼62°C), or 300 MPa at T_in=4°C (T₂∼65°C). At all T_in tested, a second pass through the HP valve (200 MPa) doubled the inactivation ratio of the total flora. Microbial patterns of raw milk were also affected; Gram-negative bacteria were less resistant than Gram-positive bacteria.     

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.     

Combined densification and pulsed electric field treatment for selective polyphenols recovery from fermented grape pomace

The aim of this study is to assess a new process for the valorization of fermented grape pomace using pulsed electric fields (PEF). The combination of densification and PEF treatment was applied on grape pomace of low relative humidity, without any addition of conductive liquid. The kinetics of extraction and the composition of polyphenols were evaluated throughout the subsequent hydro-alcoholic extraction at different temperatures.Optimal parameters of PEF treatment (field strength E = 1.2 kV·cm^− 1; energy input W = 18 kJ·kg^− 1; density ρ = 1.0 g·cm^− 3) increased the content of total polyphenols regardless of the temperature of extraction. The ratio of total anthocyanins to total flavan-3-ols at 20 °C was equal to 7.1 and 9.0 for control and PEF treated modalities, respectively. These results demonstrate the selective nature of PEF treatment in anthocyanin extraction, and thus reveal new possibilities to produce extracts with different biochemical compositions.Industrial relevanceThis study examines the feasibility of densification combined with PEF pre-treatment of relatively low humidity grape pomace for the enhancement of bioactive compounds extraction. The concentration of total phenolic compounds obtained after PEF treatment showed that the use of this technique is relevant for an industrial use, since solvent amount and extraction time can be reduced. Moreover, the selective nature of PEF opens the opportunity to produce extracts of different biochemical compositions. This process is an alternative to conventional pre-treatments of raw material (e.g. dehydration and grinding), which have impacts on product quality and are more energy consuming.     

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.     

Influence of treatment temperature on the inactivation of Listeria innocua by pulsed electric fields

The effect of treatment temperature on the bactericidal effectiveness of pulsed electric fields (PEF) applied on Listeria innocua suspended in McIllvaine buffer was investigated. Electric field intensity and number of applied pulses were applied in the ranges of 31–40 kV/cm and 5–35 pulses, respectively. Studied treatment temperatures were sustained for 10 s, and ranged between 19°C and 59°C depending on the amount of energy delivered by the PEF treatment. The application of PEF at higher temperatures proved to be more effective than either PEF at low temperatures or the applied thermal treatments by themselves. A maximum bacterial inactivation of 6-log cycles was obtained by applying either: 20 pulses of 40 kV/cm at 65°C, 25 pulses of 36 kV/cm at 61°C, or 31 pulses of 31 kV/cm at 56°C. On the other hand, a thermal treatment of 66°C sustained for 30 s reduced the bacterial population on its own by only 5-log cycles, and the application of 60 pulses of 31 kV/cm at 30°C caused only 3-log cycles of bacterial inactivation. The findings in this study suggest that PEF technology may be effectively used as an enhanced mild thermal preservation method.     

Milk enhances intestinal absorption of green tea catechins in in vitro digestion/Caco-2 cells model

The effect of milk on the absorption of polyphenols is still controversial so far. In order to determine the impact of milk addition on green tea catechins bioaccessibility and intestinal absorption an in vitro digestion/Caco-2 cell model was applied. Green tea extract (GTE) was solubilized in distilled water at 23 °C and 100 °C, combined with skimmed milk (GTE + 10% milk and GTE + 25% milk) and subjected to simulated gastric and intestinal digestion, followed by transepithelial absorption in Caco-2 cells monolayers. In the mixture with milk, gallated catechins: ECG and EGCG showed binding to milk proteins while EC and EGC seemed to have weaker affinity. Catechins were stable during gastric incubation and very sensitive to intestinal digestion. Bioaccessibility of green tea catechins brewed at 100 °C was higher than brewed at 23 °C. Catechins from digested GTE with 10% and 25% milk exhibited enhanced intestinal permeability in Caco-2 model in comparison to non-digested GTE and digested GTE without milk. Apparent permeability coefficients (P_app) of EGCG and ECG in digested GTE with 25% milk were significantly higher compared to those in GTE with 10% milk, and amounted to 2.41 × 10^− 6 cm/s and 1.39 × 10^− 6 cm/s. The recoveries of all catechins in GTE with milk in Caco-2 cells after 2 h incubation were significantly higher than that without milk. To summarize, these data suggest that milk addition may increase catechin bioavailability by enhancing their transepithelial absorption and uptake from green tea extract.     

Partial renneting of pasteurised bovine milk: Casein micelle size,heat and storage stability

The effects of partial renneting at low temperature on the casein micelle (CM) size and the storage stability of milk were investigated. Low chymosin concentrations (≤ 0.03 IMCU mL^− 1) was applied to pasteurised skim milk at 4 °C and enzyme activity was terminated by thermal application at 60 °C/3 min and 85 °C/30 min, referred to as low heat (LHT) and high heat (HHT) treatment milk, respectively. The addition of rennet with concentrations of 0.01, 0.02 and 0.03 IMCU mL^− 1 for 15 min resulted in κ-casein hydrolysis of 10, 20 and 25%, respectively. Moreover, mean CM size of milk was reduced by up to 10 nm. For LHT milk, the renneted micelles appeared to be stable for up to 17 days, especially in response to the application of 0.01 IMCU mL^− 1 and at a storage temperature of 4 °C. Severe heating at 85 °C/30 min to inactivate the enzyme caused an increase in CM size.     

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.