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.     

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.     

Effect of temperature on growth and metabolism of probiotic bacteria in milk

The growth and metabolism of six probiotic strains with documented health effects were studied in ultra-high temperature (UHT) treated milk supplemented with 0.5% (w/v) tryptone or 0.75% (w/v) fructose at different temperatures. The probiotic strains were Lactobacillus acidophilus La5, Lb. acidophilus 1748, Lb. johnsonii LA1, Lb. rhamnosus GG, Lb. reuteri SD 2112 and Bifidobacterium animalis BB12. Fermentation was followed for 48 h at 20, 30, 37 and 45 °C and the samples were analysed for pH, log cfu mL⁻¹, volatile compounds, organic acids and carbon dioxide. All six probiotic strains showed very different profiles of metabolites during fermentation, however, the two Lb. acidophilus strains were the most alike. All strains, except Lb. reuteri SD 2112, showed viable cell numbers above 6.5 log cfu mL⁻¹ after 48 h fermentation at 30, 37 and 45 °C. The probiotic strains produced different amounts of metabolic products according to temperature and fermentation time illustrating the importance of controlling these parameters.     

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.     

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.     

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.     

A combined morphologic and molecular approach for characterizing fungal microflora from a traditional Italian cheese (Fossa cheese)

Phenotypic and genotypic methods were used to identify filamentous fungi that characterize traditional Italian Fossa cheese and its ripening environment. After ageing for 60 days at a dairy, it was ripened for an additional three months in a pit. In the fully ripened cheese, moulds ranged from 3 to 3.4 log cfu g^?1 and Penicillium was the prevalent species. Pit environmental fungi ranged from 530 to 750 cfu m^?3 (air) and from 130 to 340 cfu cm^?2 (surfaces). The dominant pit strains were Alternaria spp., Aspergillus spp., Cladosporium spp. and Penicillium spp. Phylogenetic analyses of 18S rRNA gene and ITS1-5.8S-ITS2 regions highlighted Penicillium camemberti, Aspergillus nidulans and Aspergillus versicolor as traceable species occurring in both the cheese and pit environment, suggesting their involvement in the development of typical Fossa cheese characteristics. This approach may be used for the identification of microflora on other cheese varieties to better understand the fungal contribution in cheese ripening.     

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.     

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.     

Survival of probiotic bacteria in a whey cheese vector submitted to environmental conditions prevailing in the gastrointestinal tract

Various foods may be used to deliver probiotic bacteria into the gastrointestinal tract; one such example is Requeijão, a Portuguese whey cheese. Survival and stability of Bifidobacterium animalis strains BLC-1, Bb-12, and Bo, Lactobacillus acidophilus strains LAC-1 and Ki, L. paracasei ssp. paracasei strain LCS-1 and L. brevis strain LMG 6906 inoculated into Requeijão, when exposed to simulated gastrointestinal tract conditions, were assessed. Homogenates of inoculated whey cheese in 0.85% (w/v) sterile saline water were exposed to a solution of hydrochloric acid (pH 2.5–3.0) and pepsin (1000 units mL^–1) at 37 °C, and then to 0.3% (w/v) bile salts after 60 or 120 min of acid exposure. All bacterial strains retained their initial viable cell numbers. Bifidobacterium animalis strains Bb-12 and Bo, and L. brevis strain LMG 6906 exhibited the highest viable cell numbers when exposed to bile salts, whereas the other strains had variable death rates.     

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.     

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.     

Enterobacteriaceae in beef products from retail outlets in the Republic of Ireland and comparison of the presence and counts of E. coli O157:H7 in these products

This study investigated the prevalence and numbers of Enterobacteriaceae in minced beef and beef burgers purchased from supermarkets and butcher shops in the Republic of Ireland (RoI). Samples (n=1303) collected between June 2001 and April 2002 from every county in the RoI (∼60 per county) were examined for the presence of Enterobacteriaceae using method BS 5763. Overall, in the 43 beef products in which E. coli O157:H7 was present the Enterobacteriaceae counts ranged from 0.52 to 6.98 log₁₀ cfu g⁻¹. There was no correlation between the number of Enterobacteriaceae and the presence of E. coli O157:H7. There were no significant differences between Enterobacteriaceae numbers in fresh, unpackaged, minced beef samples from butcher shops and supermarkets, or in fresh, unpackaged, beef burgers from butcher shops and supermarkets. However, there were significant differences among the numbers of Enterobacteriaceae detected in different minced beef products. The numbers of Enterobacteriaceae in fresh, unpackaged, minced beef (6.54–6.98 log₁₀ cfu g⁻¹) were considerably higher than in preprepared or prepackaged minced beef (2.95–3.62 log₁₀ cfu g⁻¹).     

Encapsulation of Lactobacillus rhamnosus in double emulsions formulated with sweet whey as emulsifier and survival in simulated gastrointestinal conditions

Lactobacillus rhamnosus cultured in sweet whey and harvested in the late log phase was entrapped in the inner aqueous phase of a double water-in-oil-in-water emulsion using concentrated sweet whey as emulsifier. The primary and double emulsion droplets showed practically no changes in their morphology and droplet size with aging time. The viability of the entrapped L. rhamnosus in the double emulsion was compared to that of non-entrapped control cells exposed to low pH and bile salt conditions. The viability of the control cells (initial number = 6.57 ± 0.3 log cfu ml^?1) decreased significantly under low pH and bile salt conditions, and their survival was 71% and 89%, respectively. The survival of the entrapped cells (initial number = 6.74 ± 0.2 log cfu ml^?1) increased significantly under low pH and bile salt conditions, and their survival was 108% and 128%, respectively. It is concluded that the double emulsion protected L. rhamnosus against simulated gastrointestinal tract conditions.     

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.     

A survey of dry-salted natural casings for the presence of Salmonella spp., Listeria monocytogenes and sulphite-reducing Clostridium spores

A monitoring study was performed for the presence of Salmonella spp., Listeria monocytogenes and sulphite-reducing Clostridium spores in (internationally) traded dry-salted natural hog and sheep casings. Two hundred and fourteen consignments were examined for Salmonella spp. and L. monocytogenes, and 138 for the Clostridium spores.None of the 214 sampled consignments (25 g amounts investigated) yielded Salmonella spp., or L. monocytogenes.Differential reinforced clostridial medium was effective in detecting the presence of sulphite-reducing clostridia. Iron sulphite agar (ISA) overall showed higher clostridia counts as compared to differential reinforced clostridial agar (DRCA). The maximum spore counts obtained on DRCA and ISA were 17.5 and 2500 cfu g⁻¹, respectively. From the casings from China, 3 (of 35 hog consignments) and 7 (of 22 sheep) showed spore counts above 100 cfu g⁻¹. From the remaining 81 samples, originating from Netherlands, New Zealand and UK, none showed a count above 100 cfu g⁻¹.The relevance of the presence of sulphite-reducing Clostridium spores for the manufacture of various meat products is discussed. It is recommended that determination of the Clostridium strains present is carried out and their properties investigated in relation to the manufacture of meat products, since some of the strains may be potentially pathogenic and/or able to spoil products.     

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.     

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.     

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).     

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.