I. Fliss

Liposome encapsulated nisin Z: optimization, stability and release during milk fermentation

Liposomes prepared from different proliposomes (Pro-lipo® H, Pro-lipo® S, Pro-lipo® C and Pro-lipo® DUO) were tested for their capacity to encapsulate nisin Z. Factors affecting the entrapment process (pH and concentration of nisin Z solution and cholesterol concentration in the lipid membranes) were optimized. The use of nisin Z monoclonal antibodies made it possible to quantify, using a competitive enzyme immunoassay, and visualize, using transmission electron microscopy, nisin Z. Nisin Z was entrapped in different liposomes with encapsulation efficiencies (EE) ranging from 9.5% to 47%. The pH of nisin Z aqueous solution and nisin Z concentration had a significant effect on the amount of encapsulated nisin. An increase in cholesterol content in lipid membranes up to (20%, w/w) resulted in a slight reduction in EE. Nisin-loaded vesicles did not severely disturb Cheddar cheese fermentation and showed stability to Cheddar cheese temperature cycle. Long term stability of liposome-encapsulated nisin Z was demonstrated for 27 days at 4°C in different media including milk with different fat levels (3.25%, 2.0% and 1.0%), skim milk, sweet whey and phosphate buffer saline (PBS). Liposome stability determined as the quantity of released nisin Z was highest in milk followed by PBS and whey, respectively.

Inhibition of Listeria innocua in Cheddar Cheese by Addition of Nisin Z in Liposomes or by In Situ Production in Mixed Culture

ABSTRACT The effect of addition of purified nisin Z in liposomes to cheese milk and of in situ production of nisin Z by Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in the mixed starter on the inhibition of Listeria innocua in cheddar cheese was evaluated during 6 months of ripening. A cheese mixed starter culture containing Lactococcus lactis subsp. lactis biovar diacetylactis UL719 was selected for high-level nisin Z and acid production. Experimental cheddar cheeses were produced on a pilot scale, using the selected starter culture, from milk with added L. innocua (105 to 106 CFU/ml). Liposomes with purified nisin Z were prepared from proliposome H and added to cheese milk prior to renneting to give a final concentration of 300 IU/g of cheese. The nisin Z-producing strain and nisin Z-containing liposomes did not significantly affect cheese production and gross chemical composition of the cheeses. Immediately after cheese production, 3- and 1.5-log-unit reductions in viable counts of L. innocua were obtained in cheeses with encapsulated nisin and the nisinogenic starter, respectively. After 6 months, cheeses made with encapsulated nisin contained less than 10 CFU of L. innocua per g and 90% of the initial nisin activity, compared with 104 CFU/g and only 12% of initial activity in cheeses made with the nisinogenic starter. This study showed that encapsulation of nisin Z in liposomes can provide a powerful tool to improve nisin stability and inhibitory action in the cheese matrix while protecting the cheese starter from the detrimental action of nisin during cheese production.

Antibacterial Activities of Nisin Z Encapsulated in Liposomes or Produced In Situ by Mixed Culture during Cheddar Cheese Ripening

ABSTRACT This study investigated both the activity of nisin Z, either encapsulated in liposomes or produced in situ by a mixed starter, against Listeria innocua, Lactococcus spp., and Lactobacillus casei subsp. casei and the distribution of nisin Z in a Cheddar cheese matrix. Nisin Z molecules were visualized using gold-labeled anti-nisin Z monoclonal antibodies and transmission electron microscopy (immune-TEM). Experimental Cheddar cheeses were made using a nisinogenic mixed starter culture, containing Lactococcus lactis subsp. lactis biovar diacetylactis UL 719 as the nisin producer and two nisin-tolerant lactococcal strains and L. casei subsp. casei as secondary flora, and ripened at 7°C for 6 months. In some trials, L. innocua was added to cheese milk at 105 to 106 CFU/ml. In 6-month-old cheeses, 90% of the initial activity of encapsulated nisin (280 ± 14 IU/g) was recovered, in contrast to only 12% for initial nisin activity produced in situ by the nisinogenic starter (300 ± 15 IU/g). During ripening, immune-TEM observations showed that encapsulated nisin was located mainly at the fat/casein interface and/or embedded in whey pockets while nisin produced by biovar diacetylactis UL 719 was uniformly distributed in the fresh cheese matrix but concentrated in the fat area as the cheeses aged. Cell membrane in lactococci appeared to be the main nisin target, while in L. casei subsp. casei and L. innocua, nisin was more commonly observed in the cytoplasm. Cell wall disruption and digestion and lysis vesicle formation were common observations among strains exposed to nisin. Immune-TEM observations suggest several modes of action for nisin Z, which may be genus and/or species specific and may include intracellular target-specific activity. It was concluded that nisin-containing liposomes can provide a powerful tool to improve nisin stability and availability in the cheese matrix.

Production of antibacterial substances by bifidobacterial isolates from infant stool active against Listeria monocytogenes

Aims: This study aimed to characterize new isolates of human bifidobacteria, evaluate some of their probiotic potential and to screen these isolates for their effectiveness at inhibiting Listeria monocytogenes in vitro.

Detection of Hepatitis A Virus by the Nucleic Acid Sequence-Based Amplification Technique and Comparison with Reverse Transcription-PCR

ABSTRACT A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 × 102 PFU/ml) was obtained with NASBA, compared to 50 PFU (1 × 104PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

Increased stress tolerance of Bifidobacterium longum and Lactococcus lactis produced during continuous mixed-strain immobilized-cell fermentation.

Aims:  The effect of immobilization and long‐term continuous culture was studied on probiotic and technological characteristics of lactic acid and probiotic bacteria.

A rapid turbidometric microplate bioassay for accurate quantification of lactic acid bacteria bacteriocins

A 1 day turbidometric microplate bioassay (TMB) was developed for the rapid, accurate and precise quantification of lactic acid bacteria (LAB) bacteriocins (nisin Z and pediocin PA-1). Parameters such as the concentration of the indicator strains and the incubation time were optimized for each bacteriocin. A high correlation coefficient (r(2)=0.992+/-0.004) was obtained for the exponential regression in the nisin Z concentration range of 20-120 ng/ml with 1 x 10(7) CFU indicator strain (Pediococcus acidilactici UL5) and an incubation time of 3 h. Using these parameters, the detection limit was estimated at 80 ng/ml (3.2 IU/ml), compared to 300 ng/ml for the agar diffusion assay (ADA). High precision (<7%) and accuracy (10%) were obtained for all nisin Z concentrations tested. Similar results were obtained with pediocin PA-1 with r(2)=0.993+/-0.005, a precision (8.2%) and an accuracy lower than 15%.

In vitro inhibition activity of nisin A, nisin Z, pediocin PA-1 and antibiotics against common intestinal bacteria

Aims:  To evaluate the sensitivity of 21 common intestinal bacteria to six antibiotics and three broad‐spectrum bacteriocins (nisins Z and A and pediocin PA‐1).

Simultaneous detection and identification of hepatitis A virus and rotavirus by multiplex nucleic acid sequence-based amplification (NASBA) and microtiter plate hybridization system

Human rotavirus and hepatitis A virus (HAV) are two of the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for their detection in foodstuffs. In this study, a multiplex nucleic acid sequence-based amplification (NASBA) system was developed to detect specifically and simultaneously human rotavirus and HAV. Two sets of primers selected from published nucleic acid sequences were used in the NASBA mixture to amplify viral RNA from both viruses. Denaturing gel electrophoresis revealed two distinct RNA products with 268 and 474 nucleotides amplified from rotavirus and HAV, respectively. The specificity of the multiplex NASBA was confirmed by a microtiter plate hybridization and detection system and by Northern blot analysis using specific oligonucleotide probes. The presence of non-homologous nucleic acid and non-target microorganisms did not have any effect on the specificity of the multiplex NASBA. Using the optimized NASBA and microtiter plate hybridization conditions, as little as 400 PFU ml x (-1) of HAV and 40 PFU ml x (-1) of rotavirus were detected. The multiplex NASBA system offers advantages over monoplex virus detection systems in terms of turnaround time and cost-effectiveness.

Detection of Campylobacter jejuni in food and poultry viscera using immunomagnetic separation and microtitre hybridization

Thermophillic Campylobacter and Camp. jejuni were detected from samplesof chicken liver, gall bladder, muscle and contaminated milk and chicken meat after anenrichment step by using immunomagnetic capture of cells with monoclonal antibody againsta specific outer membrane protein of thermophilic Campylobacter. The detection ofcaptured cells was achieved using two different hybridization methods. In one of the methods,the captured cells were lysed by guanidine isothiocyanate and the 23S rRNA wasreacted with a microtitre plate‐immobilized rDNA probe specific for thermophilicCampylobacter. In the other method, the captured cells were subjected to lysis byultrasonication and the genomic DNA reacted with a microtitre plate‐immobilized RNAprobe specific for Camp. jejuni. Detection of the RNA–DNA hybrids formed in the wells was carried out using a monoclonal anti‐RNA–DNA hybrid antibody.