Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor "indicator microorganism"

BACKGROUND: Successful application of growth inhibition techniques for quantitative determination of bacteriocins relies on the sensitivity of the applied indicator microorganism to the bacteriocin to which is exposed. However, information on indicator microorganisms' performance and comparisons in bacteriocin determination with bioassays is almost non-existing in the literature. The aim of the present work was to evaluate the parameter "indicator microorganism" in bioassays carried out on solid -agar diffusion assay- and liquid -turbidometric assay- substrates, applied in the quantification of the most studied bacteriocin nisin. RESULTS: The performance of characterized microorganisms of known sources, belonging to the genera of Lactobacillus, Pediococcus, Micrococcus and Leuconostoc, has been assessed in this work in the assays of plate agar diffusion and turbidometry. Dose responses and sensitivities were examined and compared over a range of assay variables in standard bacteriocin solutions, fermentation broth filtrates and processed food samples. Measurements on inhibition zones produced on agar plates were made by means of digital image analysis. The data produced were analyzed statistically using the ANOVA technique and pairwise comparisons tests. Sensitivity limits and linearity of responses to bacteriocin varied significantly among different test-microorganisms in both applied methods, the lower sensitivity limits depending on both the test-microorganism and the applied method. In both methods, however, only two of the nine tested microorganisms (Lactobacillus curvatus ATCC 51436 and Pediococcus acidilactici ATCC 25740) were sensitive to very low concentrations of the bacteriocin and produced a linear-type of response in all kinds of samples used in this work. In all cases, very low bacteriocin concentrations, e.g. 1 IU/ml nisin, were more accurately determined in the turbidometric assay. CONCLUSION: The present work shows that in growth inhibition techniques used in bacteriocin quantification, the choice of the indicator microorganism is critical. Evaluation of sensitivity levels and type of produced responses showed that they can vary widely among different test-microorganisms and different applied methods, indicating that not all microorganisms can be used successfully as indicators and that measurements of growth inhibition in liquid media produce more reliable results

Zamjena masti u trajnim kobasicama emulzijom ekstra djevičanskog maslinovog ulja s dodatkom autohtonih bakterija mliječno-kiselog vrenja

Research background. Formulations based on vegetable or fish oil and modifications in the production technology of dry fermented sausages have emerged in recent years aiming to achieve the desirable target of reducing the fat content of these meat products. However, previous efforts have confronted many difficulties, such as high mass loss and unacceptable appearance due to intensely wrinkled surfaces and case hardening. The objective of this study is to produce and evaluate dry fermented sausages by utilising a meat protein-olive oil emulsion as fat substitute and indigenous lactic acid bacteria (LAB) with probiotic properties isolated from traditional Greek meat products. Experimental approach. A novel formulation with extra virgin olive oil and turkey protein was developed to totally replace the conventionally added pork fat. Probiotic and safety characteristics of autochthonous LAB isolates from spontaneously fermented sausages were evaluated and three LAB isolates were finally selected as starter cultures. Physicochemical, microbiological and sensory analyses were carried out in all treatments (control, Lactobacillus acidophilus, L. casei, L. sakei and Pediococcus pentosaceus) during fermentation. Results and conclusions. Ready-to-eat sausages were found to be microbiologically stable. The olive oil-based formulation produced in this study generated a mosaic pattern visible in the sliced product simulating the fat in conventional fermented sausages and was regarded as an ideal fat substitute for the production of fermented sausages. An autochthonous isolate of Lactobacillus casei adapted the best to the final products as it was molecularly identified to be present in the highest counts among the LAB isolates used as starter cultures. Novelty and scientific contribution. Α novel and high-quality dry fermented meat product was produced by replacing the added pork fat with a fat substitute based on a meat protein-olive oil emulsion. Autochthonous LAB with in vitro probiotic properties could have a potential use in large-scale novel dry fermented sausage production. Such isolates could be used as starters in an effort to standardise the production process and retain the typical organoleptic and sensory characteristics. Moreover, isolates like L. casei 62 that survived in high counts in the final products can increase the safety of fermented sausages by competing not only with pathogens but also with the indigenous microbiota and could have a potential functional value for the consumer.Pozadina istraživanja. Posljednjih godina se u proizvodnji trajnih kobasica koriste novi pripravci na bazi biljnog ili ribljeg ulja koji smanjuju udjel masti u tim mesnim proizvodima. Međutim, dosadašnji napori u dobivanju takvih proizvoda nailazili su na mnoge prepreke, kao što su veliki gubitak mase i neprihvatljiv izgled proizvoda zbog prevelike naboranosti površine i tvrdoće crijeva. Svrha je ovoga rada bila proizvesti trajne kobasice s emulzijom maslinovog ulja i proteina mesa kao zamjenom za masti, uz dodatak autohtonih bakterija mliječno-kiselog vrenja s probiotičkim svojstvima izoliranih iz tradicionalnih grčkih mesnih proizvoda, te ispitati svojstva dobivenih proizvoda. Eksperimentalni pristup. Razvijen je novi pripravak s ekstra djevičanskim maslinovim uljem i proteinima iz puretine koji bi u potpunosti zamijenio tradicionalno dodavanu svinjsku mast. Ispitana su probiotička svojstva i sigurnost primjene autohtonih bakterija mliječno-kiselog vrenja izoliranih iz tradicionalnih fermentiranih kobasica, i izdvojena su tri izolata koja su zatim upotrijebljena kao starter kulture. Tijekom fermentacije provedene su fizikalno-kemijske, mikrobiološke i senzorske analize svih ispitanih uzoraka (kontrolnog uzorka, te kobasica proizvedenih s pomoću Lactobacillus acidophilus, L. casei, L. sakei i Pediococcus pentosaceus). Rezultati i zaključci. Gotovi proizvodi bili su mikrobiološki stabilni. Dodatkom pripravka na bazi maslinovog ulja dobivena je kobasica koja je na presjeku imala mozaičan izgled sličan onom tradicionalne kobasice, pa se pripravak smatra idealnom zamjenom za mast u proizvodnji trajnih kobasica. Molekularnom je analizom utvrđeno da je među bakterijama mliječno-kiselog vrenja koje su korištene kao starter kulture u gotovom proizvodu bilo najviše bakterija Lactobacillus casei, što potvrđuje da se ta vrsta najbolje prilagođava uvjetima proizvodnje. Novina i znanstveni doprinos. Proizveden je novi visoko kvalitetni sušeni fermentirani mesni proizvod zamjenom svinjske masti emulzijom maslinovog ulja i proteina iz puretine. Autohtone bakterije mliječno-kiselog vrenja s potvrđenim probiotičkim svojstvima in vitro mogle bi se primijeniti u proizvodnji trajnih kobasica na veliko. Dobiveni bi se izolati mogli primijeniti kao starter kulture za standardizaciju postupka i očuvanje tipičnih organoleptičkih i senzorskih svojstava kobasica. Osim toga, izolati poput L. casei 62, koji su pronađeni u velikom broju u gotovom proizvodu, svojom kompetitivnošću ne samo s patogenim sojevima, već i s autohtonom mikrobiotom, povećavaju sigurnost fermentiranih kobasica te imaju potencijalnu funkcionalnu vrijednost za potrošače

Effects of Chitosan Coatings on Controlling <i>Listeria monocytogenes</i> and Methicillin-Resistant <i>Staphylococcus aureus</i> in Beef and Mutton Cuts

Ruminant meat is considered to be a potent vehicle of foodborne pathogen transmission. Edible coatings are considered to be promising for enhancing meat safety. Here, edible chitosan membranes were applied to whole cuts of beef and mutton to test the survival of the pathogenic bacteria Listeria monocytogenes and methicillin-resistant Staphylococcus aureus. Meat pieces weighing approximately 200 g were inoculated with a cocktail of three S. aureus isolates or four L. monocytogenes isolates (6.00 log10CFU/g). The meat pieces were encased in a chitosan coating formed by immersion and incubated aerobically or vacuum-packaged in LDPE/PA/LDPE bags for up to 21 days. A decrease in both S. aureus (x = −1.95 log10CFU/g, standard error = 0.23 log10CFU/g) and L. monocytogenes counts (x = −1.07 log10CFU/g, standard error = 0.26 log10CFU/g) was observed. No significant differences were observed between L. monocytogenes-spiked beef and mutton pieces; statistically higher S. aureus counts were observed in mutton versus beef under similar treatments. Aerobic storage of meat pieces inoculated with L. monocytogenes enhanced the antibacterial effects of chitosan—a trend that was not observed in meat pieces inoculated with S. aureus. According to the results, edible chitosan membranes were effective in controlling the growth of S. aureus and L. monocytogenes

Effects of Chitosan Coatings on Controlling Listeria monocytogenes and Methicillin-Resistant Staphylococcus aureus in Beef and Mutton Cuts

Ruminant meat is considered to be a potent vehicle of foodborne pathogen transmission. Edible coatings are considered to be promising for enhancing meat safety. Here, edible chitosan membranes were applied to whole cuts of beef and mutton to test the survival of the pathogenic bacteria Listeria monocytogenes and methicillin-resistant Staphylococcus aureus. Meat pieces weighing approximately 200 g were inoculated with a cocktail of three S. aureus isolates or four L. monocytogenes isolates (6.00 log10CFU/g). The meat pieces were encased in a chitosan coating formed by immersion and incubated aerobically or vacuum-packaged in LDPE/PA/LDPE bags for up to 21 days. A decrease in both S. aureus (x = &minus;1.95 log10CFU/g, standard error = 0.23 log10CFU/g) and L. monocytogenes counts (x = &minus;1.07 log10CFU/g, standard error = 0.26 log10CFU/g) was observed. No significant differences were observed between L. monocytogenes-spiked beef and mutton pieces; statistically higher S. aureus counts were observed in mutton versus beef under similar treatments. Aerobic storage of meat pieces inoculated with L. monocytogenes enhanced the antibacterial effects of chitosan&mdash;a trend that was not observed in meat pieces inoculated with S. aureus. According to the results, edible chitosan membranes were effective in controlling the growth of S. aureus and L. monocytogenes

Effect of Chitosan and Alginate-Based Edible Membranes with Oregano Essential Oil and Olive Oil in the Microbiological, Physicochemical and Organoleptic Characteristics of Mutton

Edible chitosan or alginate coatings and their combinations with oregano essential oil or olive oil, have been examined for their effect on the microbiological, physicochemical and organoleptic characteristics of mutton. The results indicated that these edible coatings can contribute to maintaining good quality characteristics and extending mutton shelf-life. The total mesophilic counts in mutton ranged from 3.48 to 8.00 log10 CFU/g, the total psychrophilic counts from 4.00 to 9.50 log10 CFU/g, the B. thermosphacta counts from 2.30 to 7.77 log10 CFU/g and the lactic acid bacteria counts from 2.00 to 5.85 log10 CFU/g. Chitosan coatings significantly (p 10 cfu/g), the B. thermosphacta and the lactic acid bacteria counts in mutton. Alginate exhibited a lower L* value and a higher a* value and chroma compared with the control and chitosan lots. No significant differences were observed in the chemical composition of meat pieces among the experimental groups. Oregano oil positively affected the sensory attributes of meat. The most favourable combination, based on the microbiological counts, the organoleptic characteristics and the shelf-life extension of mutton, was that of chitosan with oregano essential oil

Effect of olive leaf (Olea europea L.) extracts on protein and lipid oxidation of long-term frozen n-3 fatty acids-enriched pork patties

Our previous study has demonstrated the protective effects of olive leaf extracts on the oxidation of pork patties from n-3 fatty acid-enriched meat during refrigerated storage. The target of the present study was to examine these effects during frozen storage. Results showed that frozen storage accelerated (P. =. 0.05) both lipid and protein oxidation in pork patties, but an addition of olive leaf extract at 200. mg gallic acid equivalent/kg improved sensory attributes by delaying oxidation of lipids (reduction (P. =. 0.05) of conjugated dienes, hydroperoxides and malondialdehyde), and of proteins (reduction (P. =. 0.05) of protein carbonyls and inhibition (P. =. 0.05) of the decrease of protein sulfhydryls). © 2014