Effect of acidification on the activity of probiotics in yoghurt during cold storage

The viability of Lactobacillus acidophilus LAFTI^® L10, Bifidobacterium lactis LAFTI^® B94, and L. paracasei LAFTI^® L26 and their proteolytic activities were assessed in yoghurt at different termination pH of 4.45, 4.50, 4.55, and 4.60 in the presence of L. delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342 during 28 days of storage at 4 °C. All strains achieved the recommended level of 6.00 log cfu g⁻¹ of the product with L. acidophilus LAFTI^® L10 and L. paracasei LAFTI^® L26 exceeding the number to 8.00 and 7.00 log cfu g⁻¹, respectively. Lactobacilli strains showed a good cellular stability maintaining constant concentration throughout storage period regardless of termination pH. On the other hand, the cell counts of B. lactis LAFTI^® B94 decreased by one log cycle at the end of storage. The presence of probiotic organisms enhanced proteolysis significantly in comparison with the control batch containing L. delbrueckii ssp. bulgaricus Lb1466 and S. thermophilus St1342 only. The proteolytic activity varied due to termination pH, but also appeared to be strain related. The increased proteolysis improved survival of L. delbrueckii ssp. bulgaricus Lb1466 during storage resulting in lowering of pH and production of higher levels of organic acids, which might have caused the low cell counts for B. lactis LAFTI^® B94.     

Proteolytic pattern and organic acid profiles of probiotic Cheddar cheese as influenced by probiotic strains of Lactobacillus acidophilus,Lb. paracasei,Lb. casei or Bifidobacterium sp.

Cheddar cheeses were produced with starter lactococci and Bifidobacterium longum 1941, B. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. paracasei LAFTI^® L26, Lb. acidophilus 4962 or Lb. acidophilus LAFTI^® L10 to study the survival of the probiotic bacteria and the influence of these organisms on proteolytic patterns and production of organic acid during ripening period of 6 months at 4 °C. All probiotic adjuncts survived the manufacturing process of Cheddar cheese at high levels without alteration to the cheese-making process. After 6 months of ripening, cheeses maintained the level of probiotic organisms at >8.0 log₁₀ cfu g⁻¹ with minimal effect on moisture, fat, protein and salt content. Acetic acid concentration was higher in cheeses with B. longum 1941, B. lactis LAFTI^® B94, Lb. casei 279 and Lb. paracasei LAFTI^® L26. Each probiotic organism influenced the proteolytic pattern of Cheddar cheese in different ways. Lb. casei 279 and Lb. paracasei LAFTI^® L26 showed higher hydrolysis of casein. Higher concentrations of free amino acids (FAAs) were found in all probiotic cheeses. Although Bifidobacterium sp. was found to be weakly proteolytic, cheeses with the addition of those strains had highest concentration of FAAs. These data thus suggested that Lb. acidophilus 4962, Lb. casei 279, B. longum 1941, Lb. acidophilus LAFTI^® L10, Lb. paracasei LAFTI^® L26 and B. lactis LAFTI^® B94 can be applied successfully in Cheddar cheese.     

Probiotic properties of folate producing Streptococcus thermophilus strains

This study aimed at assessing the probiotic potential of two high folate producing Streptococcus thermophilus strains (RD102 and RD104) isolated from Indian fermented milk products by both in vitro and in vivo tests. These strains were able to survive at pH 2.5 and 2% bile with a good bile salt hydrolase activity, cell surface hydrophobicity and sensitivity to most of the clinically important antibiotics. On evaluation for gastrointestinal transit tolerance these showed a viable count of 5 log cfu mL^?1 and 7 log cfu mL^?1, respectively in simulated gastrointestinal juice of pH 2.0 and 2% bile. During the in vivo feeding trial in mice the strains showed a viable count of about 7 log cfu g^?1 faeces and 6 log cfu g^?1 of large intestine, respectively. These strains were hence observed to possess favorable strain specific probiotic properties and have the potential to be a source of novel probiotics.     

Survival and activity of selected probiotic organisms in set-type yoghurt during cold storage

The growth and metabolism of two probiotic organisms (L. acidophilus LAFTI^® L10 and Lactobacillus casei LAFTI^® L26) and a regular yoghurt culture (L. delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342) were studied in yoghurt containing 0.5%, 1.0%, and 1.5% (w/v) of high amylose corn starch powder (Hi-maize^®) or inulin. Viable cell counts of probiotic organisms, their metabolites and proteolytic activities, and viscosity of the yoghurts were determined during refrigerated storage for 28 d at 4 ^oC. In the presence of inulin, cultures showed better retention of viability (8.0 log cfu g⁻¹) in comparison with that of Hi-maize, which had a reduction by one log cycle. Lower concentrations of 0.5–1.0% Hi-maize improved (P<0.05) the production of propionic acid and also increased proteolytic activity of probiotic organisms substantially. A greater release of free amino acids may have sustained better growth of the organisms in yoghurts. Supplementation with either Hi-maize or inulin increased the viscosity of probiotic yoghurts significantly (P<0.05).     

The influence of multi stage alginate coating on survivability of potential probiotic bacteria in simulated gastric and intestinal juice

The probiotics, Lactobacillus acidophilus PTCC1643 and Lactobacillus rhamnosus PTCC1637, were encapsulated into uncoated calcium alginate beads and the same beads were coated with one or two layers of sodium alginate with the objective of enhancing survival during exposure to the adverse conditions of the gastro-intestinal tract. The survivability of the strains, was expressed as the destructive value (decimal reduction time). Particle size distribution was measured using laser diffraction technique. The thickness of the alginate beads increased with the addition of coating layers. No differences were detectable in the bead appearance by scanning electron microscopy (SEM). The alginate coat prevented acid-induced reduction of the strains in simulated gastric juice (pH 1.5, 2 h), resulting in significantly (P < 0.05) higher numbers of survivors. After incubation in simulated gastric (60 min) and intestinal juices (pH 7.25, 2 h), number of surviving cells were 6.5 log cfu mL^?1 for L. acidophilus and 7.6 log cfu mL^?1 for L. rhamnosus by double layer coated alginate microspheres, respectively, while 2.3 and 2.0 log cfu mL^?1 were obtained for free cells, respectively.     

Incorporation of selected nutraceuticals and probiotic bacteria into a fermented milk

Yoghurts were produced from a base milk containing three important nutraceuticals, namely ω-3-fatty acids, isoflavones and phytosterols. The cultures employed to make the yoghurts were single probiotic strains of Lactobacillus gasseri or Bifidobacterium infantis and, to achieve a short production time, a two-stage fermentation procedure was used with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus providing the rapid acidification. Yoghurts containing counts of >1.0×10⁸ cfu mL⁻¹ of the individual probiotics and high counts of the traditional species from yoghurt were awarded overall scores for sensory acceptability >4.0 out of 5.0; the nutraceuticals appeared to have no adverse effect on flavour. Storage trials at 5 °C showed that the viability of the probiotic cultures was retained over 15 days.     

Effect of combinations of high-pressure treatment and bacteriocin-producing lactic acid bacteria on the survival of Listeria monocytogenes in raw milk cheese

The combined effect of high-pressure (HP) treatment and bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Listeria monocytogenes Scott A in cheeses made from raw milk that was inoculated with the pathogen at 4.80 log cfu mL⁻¹, a commercial starter and one of seven strains of BP-LAB was investigated. On day 3, the counts of L. monocytogenes were 7.03 log cfu g⁻¹ in a control cheese (without BP-LAB, not HP treated), 6.06–6.74 log cfu g⁻¹ in cheeses with BP-LAB, 6.13 log cfu g⁻¹ in a cheese without BP-LAB and treated on day 2 at 300 MPa, 2.01 log cfu g⁻¹ in a cheese without BP-LAB and treated on day 2 at 500 MPa, 3.83–5.43 log cfu g⁻¹ in cheeses with BP-LAB and treated on day 2 at 300 MPa, and 1.81 log cfu g⁻¹ or less in cheeses with BP-LAB and treated on day 2 at 500 MPa. HP treatment was more effective on day 51 than on day 2.     

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.     

Production of traditional Greek yoghurt using Lactobacillus strains with probiotic potential as starter adjuncts

Lactobacillus plantarum ACA-DC 146 and L. paracasei subsp. tolerans ACA-DC 4037 were examined for their potential application as adjuncts in the production of traditional Greek set-type yoghurt. Both strains displayed low milk acidification activity, while no inhibition was observed towards or from the yoghurt starters used. Yoghurt produced with L. paracasei subsp. tolerans ACA-DC 4037 exhibited the best sensory properties, with a rich traditional smooth taste, and the strain was selected for further trials. Yoghurt produced with this strain as an adjunct had good physicochemical properties. After 2 weeks of refrigerated storage, microbial loads (>7.0 log cfu g⁻¹) were in accordance with international recommendations and guidelines for probiotic and starter cultures in milk products. Increasing the microbial load further, using concentrated and encapsulated inocula (10–11 log cfu g⁻¹), gave yoghurt with long fermentation times and poor organoleptic properties.     

Influence of probiotic bacteria on the proteolysis profile of a semi-hard cheese

Two probiotic strains, Lactobacillus acidophilus and Lactobacillus paracasei subsp. paracasei, were used as adjunct cultures in semi-hard cheesemaking experiments, in order to study their influence on proteolysis during ripening. Cheeses with and without probiotic bacteria were manufactured. The population of probiotics remained above 10⁷ cfu g⁻¹ during all ripening, and they did not influence primary proteolysis. However, L. acidophilus produced a significant increase in the level of low molecular weight nitrogen compounds and individual free amino acids; the amino acid profiles were also different. Multivariate analysis of peptide profiles showed that samples were grouped mainly by ripening time, although the impact of probiotics was also noticeable. L. acidophilus showed a clear influence on secondary proteolysis, while a minor effect of L. paracasei was evidenced at the end of the ripening. These results showed that the tested strains influenced distinctly proteolysis of cheeses, probably as a consequence of their different proteolytic systems and their activity via the alimentary matrix (cheese).     

Improving the viability of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 in white-brined cheese by microencapsulation

The viability of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 microencapsulated by either an extrusion or an emulsion technique and used in white-brined cheese was monitored. Both microencapsulation techniques were effective in keeping the numbers of probiotic bacteria higher than the level of the therapeutic minimum (>10⁷ cfu g^?1). While the counts of probiotic bacteria decreased approximately 3 log in the control cheese in which probiotics were used as free cells, the decrease was more limited in the cheeses containing microencapsulated cells (approximately 1 log). Medium- and long-chain free fatty acid contents of the cheeses with immobilized probiotics were much higher than in the control cheese. Similarly, cheeses made with immobilized probiotics contained higher acetaldehyde and diacetyl levels than the control. Experimental cheeses containing microencapsulated probiotics were not different from the control cheese in terms of sensory properties.     

Characterization of bacteriocin ST8KF produced by a kefir isolate Lactobacillus plantarum ST8KF

Lactobacillus plantarum ST8KF, isolated from kefir, produced a 3.5 kDa bacteriocin (bacST8KF) active against Lb. casei, Lb. salivarius, Lb. curvatus and Listeria innocua. BacST8KF was sensitive to proteolytic enzymes, but stable between pH 2.0 and 10.0, and heat resistant (20 min at 121 °C). BacST8KF did not adsorb to the surface of the producer cell. Maximum activity (25,600 AU mL⁻¹) was recorded in MRS broth with glucose, in MRS broth with glucose replaced by sucrose, and in MRS broth with glucose, supplemented with KH₂PO₄ after 24 h at 30 °C. Tri-ammonium citrate and glycerol in excess of 5.0 g L⁻¹ repressed bacST8KF production. Production of bacST8KF increased from 800 AU mL⁻¹ after 3 h of fermentation in MRS broth at 30 °C to 12,800 AU mL⁻¹ after 9 h and to 51,200 AU mL⁻¹ after 27 h. These results suggest that bacST8KF may be a secondary metabolite and shows that its mode of activity is bacteriostatic.     

Assessing the proteolytic and lipolytic activities of single strains of mesophilic lactobacilli as adjunct cultures using a Caciotta cheese model system

Seventeen strains of mesophilic lactic acid bacteria, isolated from cheese (non-starter lactic acid bacteria, NSLAB) or sourdough, were used individually as adjunct cultures in a Caciotta cheese model system. Adjunct cultures were monitored by randomly amplified polymorphic DNA analysis and their cell counts mainly varied from ca. 9.0 to 8.0 log cfu g⁻¹ throughout 36 days of ripening. Adjunct cultures influenced differently cheese proteolysis. Both NSLAB and sourdough strains caused an extensive secondary proteolysis; however, some NSLAB strains produced the highest concentration of free amino acids. Principal component analysis (PCA) differentiated cheeses manufactured with NSLAB strains Lactobacillus parabuckneri B₉F_ST, Lb. paracasei B₆₁F₅, Lb. curvatus 2768 and Lb. rhamnosus ATCC 7469 based on the accumulation of Lys, Glu, Phe, Hist, Asp and Met. Assessment of cheese lipolysis showed that: (i) highest concentrations of free fatty acids (FFA) were found with NSLAB strains Lb. rhamnosus ATCC 7469 and Lb. casei subsp. pseudoplantarum 2742 (ca. 10 500 mg kg⁻¹); (ii) PCA differentiated cheeses manufactured with NSLAB strains Lb. rhamnosus ATCC 7469 and Lb. casei subsp. pseudoplantarum 2742 based on the accumulation of palmitic (C16:0) and linoleic (C18:2) acids, and those with Lb. curvatus 2768 and Lb. parabuckneri B₉F_ST based on the high concentration of short chain FFA; (iii) the cheese made with sourdough strain Lb. sanfranciscensis CB1 had the highest levels of unsaturated FFA.     

Microbiological conditions of moisture-enhanced pork before and after cooking

Substantial numbers of aerobic bacteria but few coliforms or Listeria spp. and no Escherichia coli were recovered from both swab samples and brines circulated in cleaned equipment used for injecting pork loins. After meat was processed for 30 or 60 min, the numbers of aerobic bacteria in brines had increased by >1 log unit, to about 4.5 log cfu ml⁻¹, but coliforms were <2 and E. coli and Listeria spp. were <1 log cfu ml⁻¹. The numbers of bacteria on the surfaces of pork loins before and after injection of the meat were similar. No bacteria were recovered from the deep tissues of the uninjected meat, but aerobic bacteria were recovered at log-mean numbers of 2.1 log cfu g⁻¹ and coliforms at log-total numbers of 1.2 log cfu 25 g⁻¹ from 25 samples of deep tissues of injected meat. Aerobic bacteria were recovered at log total numbers of 1.0 log cfu 25 g⁻¹ from 25 samples of injected pork cooked to a central temperature of 61 °C, but no bacteria were recovered from the deep tissues of meat cooked to 70 °C. The findings suggest that moisture-enhanced pork cooked to a medium rare condition can be microbiologically safe.     

Fresh-cheesemilk formulation fermented by a combination of freeze-dried citrate-positive cultures and exopolysaccharide-producing lactobacilli with liquid lactococcal starters

Milk formulation (4% fat and 5% protein) prepared to simulate fresh cheese production was inoculated with: (1) 10⁷ cfu mL⁻¹ of fresh liquid starters of Lactococcus lactis ssp. lactis T1 and Lc. lactis ssp. cremoris T2, (2) a freeze-dried exopolysaccharide-producing (EPS) strain of Lactobacillus rhamnosus RW-9595M, and (3) freeze-dried Leuconostoc cremoris LM057 or Lc. lactis ssp. lactis var. diacetylactis MD089 strains. The effect of inoculation rate of the freeze-dried starters (between 10⁶ and 10⁷ cfu mL⁻¹) and incubation temperature (between 23.5 and 36.5 °C) on evolution of pH and the various populations during fermentation was examined. Texture (apparent viscosity, syneresis potential) and chemical composition (diacetyl, acetaldehyde) of the fermented milks were also determined. Milk was incubated until a pH of 4.6 was obtained, which required between 6 and 10 h depending on temperature.In the range of inoculation levels used, there was no significant effect of the presence of lactobacilli, Ln. cremoris or Lc. lactis ssp. lactis var. diacetilactis on the growth of the lactococci. There was a direct correlation between the inoculation rates of the freeze-dried cultures and their final populations in the fermented milks. The growth of the cultures were also affected by temperature, Ln. cremoris growing less as incubation temperature increased, while the opposite was noted with Lb. rhamnosus. The apparent viscosity of the fermented milk was significantly affected by incubation temperature, but there was no correlation between apparent viscosity and the final population in lactobacilli. Of the three variables studied, the highest correlation with diacetyl content was obtained with the inoculation level of the Leuconostoc strain.     

Microbiological,compositional, biochemical and textural characterisation of Caciocavallo Pugliese cheese during ripening

A microbiological, compositional, biochemical and textural characterisation of the pasta filata Caciocavallo Pugliese cheese during ripening is reported. Fully ripened cheese contained a total of ca. log 8.0 cfu g⁻¹ mesophilic bacteria and ca. log 6.0 cfu g⁻¹ presumptive staphylococci, while the number of thermophilic and mesophilic rod and coccus lactic acid bacteria varied during ripening. A two-step RAPD-PCR protocol was used to differentiate biotypes. The natural whey starter was composed mainly of Lactobacillus delbrueckii, Lb. fermentum, Lb. gasseri, Lb. helveticus and Streptococcus thermophilus strains. After day 1 of ripening, Lb. delbrueckii became dominant and some strains of Enterococcus durans and E. faecalis appeared. Non-starter lactic acid bacteria, such as Lb. parabuchneri and Lb. paracasei subsp. paracasei formed a large part of the lactic microflora at 42 and 60 d of ripening. The level of pH 4.6-soluble nitrogen increased from the outer to the inner of the cheese and also increased in each section as ripening progressed, attaining values of 18–15%. Urea-PAGE electrophoresis showed that degradation of α_s1-casein was more rapid than that of β-casein throughout ripening and the rates at which both caseins were degraded greatly increased from the outside to the inside of the cheese. Based on the primary proteolysis products, both chymosin and plasmin appeared to be active. RP-HPLC profiles of the 70% ethanol-soluble, pH 4.6-soluble nitrogen, showed a large number of peaks, indicating a heterogeneous mixture of proteolytic products. There were both age- and section-related changes in the area of the different peptide peaks. Butyric (C4:0), caproic (C6:0), palmitic (C16:0) and oleic (C18:1) acids were the free fatty acids found at the highest concentrations. The level of short chain fatty acids (e.g., butyric and caproic) decreased from the middle and inner to outer sections of the cheese. Peptidase activity in the curd was pronounced, increased during ripening and varied with the cheese section. The greatest increase of the peptidase activity coincided with a change in the lactic microflora and with the prevalence of non-starter lactic acid bacteria. Microbial esterases were supposed to be active together with rennet paste. Little change in the firmness and fractures stress during maturation were found by textural analyses of the raw cheese. The flowability was similar to that of typical low-moisture Mozzarella cheese, while stretchability was lower. The heat-induced changes in phase angle of Caciocavallo Pugliese cheese indicated a phase transition from largely elastic rheological characteristics in unheated cheese to a more viscous and fluid character in melted cheese.     

The protective effect of monosodium glutamate on survival of Lactobacillus rhamnosus GG and Lactobacillus rhamnosus E-97800 (E800) strains during spray-drying and storage in trehalose-containing powders

The use of trehalose as a means of preserving Lactobacillus rhamnosus GG (LGG) and L. rhamnosus E-97800 (E800) during spray-drying and the effects of incorporated monosodium glutamate (MSG) in the carrier medium on the survival rates during drying and storage were examined. E800 was more resistant to heat than LGG in 20%, w/w, trehalose; the d-values at 65 °C were 14 s and 5.1 s, respectively. An air outlet temperature of 65–70 °C was taken as optimal for the drying process, as the resultant moisture levels in trehalose containing these bacteria were 4.1% (w/w) and 3.79% (w/w) with corresponding viable counts of 3.65 × 10⁸ cfu mL^?1 and 1.80 × 10⁹ cfu mL^?1, respectively. The presence of MSG increased the final viable counts of LGG and E800 to 3.05 × 10⁹ cfu mL^?1 and 1.30 × 10⁹ cfu mL^?1, respectively. Survival of LGG and E800 remained constant at a minimum level of ～10⁸ cfu mL^?1 during storage at 25 °C in trehalose–MSG medium.     

Safety aspects of probiotic bacterial strains Lactobacillus rhamnosus HN001 and Bifidobacterium animalis subsp. lactis HN019 in human infants aged 0–2 years

Given the relatively immature state of the neonatal gut and gut-associated immune system, the safety of probiotic strains for use as ingredients in infant milk formulae must be demonstrated in infant populations. As part of a double-blind placebo-controlled clinical trial of two commercially available probiotic strains in the reduction of risk for infant eczema, a number of safety outcomes were measured. Infants received daily doses of Lactobacillus rhamnosus HN001 (6 × 10⁹ cfu day^?1) or Bifidobacterium animalis subsp. lactis HN019 (9 × 10⁹ cfu day^?1), or placebo from birth to 24 months. Mothers received the same treatment from 35 weeks gestation, for up to 6 months postnatally while breastfeeding. No statistically significant differences were observed between the treatment groups for study withdrawal, incidence of adverse events, morphometric data, wheeze, and antibiotic use over the treatment period. We conclude that probiotics strains HN001 and HN019 were safe and well tolerated in infants, and did not affect normal growth.     

Kinetic parameters of Escherichia coli O157:H7 survival during fermentation of milk and refrigeration of home-made yoghurt

The survival parameters of Escherichia coli O157:H7 during milk fermentation (carried out by the LIM or “longer incubation method” at 30 °C, or by the SIM or “short incubation method” at 43 °C) and storage of home-made yoghurt at refrigeration temperatures (2, 4, or 8 °C) were studied. The E. coli O157:H7 counts increased slightly during fermentation by the LIM, from 5.1 to 5.4 log cfu mL⁻¹, and it was not found after 21 d of storage at 2 or 4 °C, and after 10 d at 8 °C. The microorganism counts increased from 4.8 to 5.4 log cfu mL⁻¹ during the SIM, and it was not detected after 7 d stored at 8 °C. The microorganism grew faster at 43 °C (generation time=0.93 h) than at 30 °C (4.12 h) during the fermentation period. The death time decreased with the increase of the storage temperature (from 38.1 h at 2 °C to 30.1 h at 8 °C) in the yoghurt produced by fermentation at 30 °C; however, a clear relationship between death time and storage temperature was not evident at 43 °C. The pH values of the yoghurt ranged from 4.0 to 4.7.     

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.