Diversity of stress tolerance in Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum: A multivariate screening study

Sixty-three strains of the taxonomically related species Lactobacillus plantarum subsp. plantarum, L. plantarum subsp. argentoratensis, L. paraplantarum and L. pentosus isolated from sourdoughs and other food and non-food sources and 14 strains of other members of the genus Lactobacillus were screened for their tolerance of acid, alkaline, heat, oxidative, osmotic, detergent and starvation stresses in order to evaluate the diversity of stress response. Most strains of the L. plantarum group were highly tolerant of acid, alkaline and osmotic stress and highly sensitive to detergent stress, while a larger diversity was found for other stress. Multivariate analysis allowed grouping the strains in clusters with similar response patterns. Stress response patterns in the L. plantarum group were similar to those of species of the L. casei/L. paracasei group but clearly different from those of other mesophilic Lactobacillus. No relationship was found between grouping obtained on the basis of stress response patterns and by genotypic fingerprinting (rep-PCR), nor with the taxonomic position or isolation source of the strains. Further experiments with selected strains showed that exponential phase cells were generally but not always more sensitive than stationary phase cells. The ability to grow under stressful conditions showed a slightly better correlation with the ecological conditions prevailing in the isolation niches of the strains.This study will be the basis for further investigations to identify and exploit the basis of diversity in the stress response of lactic acid bacteria.     

Antioxidant activity of Spirulina platensis extracts by supercritical carbon dioxide extraction

Supercritical CO₂ extraction of antioxidants from Spirulina platensis was optimized using response surface methodology. About 10.26 g/kg of extracts from S. platensis could be obtained under the optimum conditions of 48 °C at 20 MPa over a 4 h period. The antioxidant activity of the extracts prepared under the optimized condition, determined by linoleic acid peroxidation inhibition method, was lower compared with BHT and Trolox, but significantly higher than α-tocopherol in 300 min and became similar to α-tocopherol after that. The components of the extracts were further analyzed, and the results showed that the extracts contained 85.1 g/kg of flavonoids, 77.8 g/kg of β-carotene, 113.2 g/kg of vitamin A and 3.4 g/kg of α-tocopherol, which may contribute greatly to their high antioxidant activity. The main fatty acids in the extracts were palmitic acid (35.32%), linolenic acid (21.66%) and linoleic acid (20.58%).     

Binding of mutagens to exopolysaccharide produced by Lactobacillus plantarum mutant strain 301102S

Exopolysaccharide (EPS) was produced by Lactobacillus plantarum 301102 on exposure to the mutagenic action of acridine orange and novobiocin. The biological characteristics of this mutant strain 301102S were the same as those of the parent strain, but fermented milk prepared with the mutant strain showed antimutagenic activity on 3-amino-1,4-dimethyl-5H-pyrido indole. Only EPS-bound cells of strain 301102S showed binding ability to mutagens such as heterocyclic amines, and the mutagens were inactivated by binding to EPS. The binding ability was affected by pH; the greatest percentage binding was noted at pH 8.0. Addition of Mg²⁺ and sodium dodecyl sulfate, but not oxgall, inhibited the binding ability. Therefore, the binding mechanism of the EPS may consist of ion-exchange and hydrophobic bonds, and the EPS would bind mutagens in the intestine.     

The effect of Lactobacillus buchneri and Lactobacillus plantarum on the fermentation, aerobic stability, and ruminal degradability of low dry matter corn and sorghum silages

The effect of Lactobacillus buchneri, alone or in combination with Lactobacillus plantarum, on the fermentation, aerobic stability, and ruminal degradability of low dry matter corn and sorghum silages was studied under laboratory conditions. The inoculants were applied at 1 x 10(6) cfu/g. Silages with no additives served as control. After treatment, the chopped forages were ensiled in 1.5-L anaerobic jars. Three jars per treatment were sampled on d 2, 4, 8, 15, and 90. After 90 d of storage, the silages were subjected to an aerobic stability test lasting 5 d, in which CO2 production, as well as chemical and microbiological parameters, was measured to determine the extent of aerobic deterioration. At the end of the ensiling period (d 90), the L. buchneri- and L. buchneri + L. plantarum-inoculated silages had significantly higher levels of acetic acid than the control and L. plantarum-inoculated silages. Therefore, yeast activity was impaired in the L. buchneri- and L. buchneri + L. plantarum-inoculated silages. As a result, L. buchneri, alone or in combination with L. plantarum, improved aerobic stability of the low dry matter corn and sorghum silages. The combination of L. buchneri and L. plantarum reduced ammonia N concentrations and fermentation losses in the silages compared with L. buchneri alone. However, L. buchneri, L. plantarum, and a combination of L. buchneri + L. plantarum did not effect in situ rumen dry matter, organic matters, or neutral detergent fiber degradability of the silages. The L. buchneri was very effective in protecting the low dry matter corn and sorghum silages exposed to air under laboratory conditions. The use of L. buchneri, alone or in combination with L. plantarum, as a silage inoculant can improve the aerobic stability of low dry matter corn and sorghum silages by inhibition of yeast activity.     

Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses

Ninety-eight Lactobacillus plantarum strains isolated from Italian and Argentinean cheeses were evaluated for probiotic potential. After a preliminary subtractive screening based on the presence of msa and bsh genes, 27 strains were characterized. In general, the selected strains showed high resistance to lysozyme, good adaptation to simulated gastric juice, and a moderate to low bile tolerance. The capacity to agglutinate yeast cells in a mannose-specific manner, as well as the cell surface hydrophobicity was found to be variable among strains. Very high β-galactosidase activity was shown by a considerable number of the tested strains, whereas variable prebiotic utilization ability was observed. Only tetracycline resistance was observed in two highly resistant strains which harbored the tetM gene, whereas none of the strains showed β-glucuronidase activity or was capable of inhibiting pathogens. Three strains (Lp790, Lp813, and Lp998) were tested by in vivo trials. A considerable heterogeneity was found among a number of L. plantarum strains screened in this study, leading to the design of multiple cultures to cooperatively link strains showing the widest range of useful traits. Among the selected strains, Lp790, Lp813, and Lp998 showed the best probiotic potential and would be promising candidates for inclusion as starter cultures for the manufacture of probiotic fermented foods.     

Production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis) with bacteriocinogenic strains of Lactobacillus plantarum and Lactobacillus curvatus

Lactobacillus plantarum 423, producer of bacteriocin 423, Lactobacillus curvatus DF38, producer of curvacin DF38, and a bacteriocin-negative mutant of L. plantarum 423 (423m) were evaluated as starter cultures in the production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis). Growth of L. plantarum 423 and L. curvatus DF38 was best supported in Blesbok salami, as revealed by the highest growth rate during sweating, cold smoking and maturation, and final cell numbers after 70 days (1 × 10⁸ and 5 × 10⁷ cfu/g, respectively). Growth of Listeria innocua was the best suppressed in Blesbok salami fermented with L. plantarum 423 and L. curvatus DF38. Growth of L. innocua in horse salami was best suppressed when fermented with L. curvatus DF38. The final pH of salami fermented with L. plantarum 423 and L. plantarum 423m was slightly lower (4.4) compared to the pH of salami produced with L. curvatus DF38 (pH 4.7). No significant differences (P > 0.05) were recorded by a trained sensory taste panel amongst the three starter cultures regarding colour and venison like aroma. Horse, Blesbok and Springbok salami were rated significantly higher (P ? 0.05) in salami flavour than mutton salami, which was rated the lowest for this attribute. Blesbok salami was rated the highest for sour meat aroma, while beef salami was rated the lowest. Springbok salami was rated the highest in terms of oily mouth feel. Beef salami had the most compact structure and horse salami the softest structure of all meat types fermented. In general, salami produced with L. plantarum 423 yielded the best sour meat aroma, colour, texture, venison like flavour, sour meat flavour and oily mouthfeel and is considered superior to the L. plantarum mutant (strain 423m) and L. curvatus DF38.     

Comparison of essential oil compositions of Salvia mirzayanii obtained by supercritical carbon dioxide extraction and hydrodistillation methods

Essential oil of Salvia mirzayanii cultivated in Iran was obtained by hydrodistillation and supercritical (carbon dioxide) extraction methods. The oil was analysed by capillary gas chromatography using flame ionization and mass spectrometric detections. The compounds were identified according to their retention indices and mass spectra (EI, 70 eV). The effects of different parameters such as pressure, temperature, modifier volume and extraction times (dynamic and static) on the supercritical fluid extraction (SFE) of S. mirzayanii oil were investigated. The results showed that, under a pressure of 35.5 MPa, temperature of 35 °C, 6% methanol, dynamic extraction time of 50 min and static extraction time of 30 min, extraction was more selective for the linalyl acetate. Thirty four compounds were identified in the hydrodistilled oil. The major components of S. mirzayanii were linalyl acetate (7.6%), 1,8-cineole (8.0%), linalool (9.0%) and 8-acetoxy linalool (11.0%). However, by using supercritical carbon dioxide in optimum conditions, only three components contain more than 63% of the oil. The yield of the obtained oil based on hydrodistillation was 2.20% (v/w). Extraction yield based on the SFE varied in the range of 1.50–9.67% (w/w) under different conditions. The results revealed that, in Iranian S. mirzayanii oil, linalyl acetate is a major component.     

Survival of Lactobacillus plantarum in model solutions and fruit juices

The aim of the work was to study the survival of Lactobacillus plantarum NCIMB 8826 in model solutions and develop a mathematical model describing its dependence on pH, citric acid and ascorbic acid. A Central Composite Design (CCD) was developed studying each of the three factors at five levels within the following ranges, i.e., pH (3.0-4.2), citric acid (6-40 g/L), and ascorbic acid (100-1000 mg/L). In total, 17 experimental runs were carried out. The initial cell concentration in the model solutions was approximately 1 × 10⁸ CFU/mL; the solutions were stored at 4 °C for 6 weeks. Analysis of variance (ANOVA) of the stepwise regression demonstrated that a second order polynomial model fits well the data. The results demonstrated that high pH and citric acid concentration enhanced cell survival; one the other hand, ascorbic acid did not have an effect. Cell survival during storage was also investigated in various types of juices, including orange, grapefruit, blackcurrant, pineapple, pomegranate, cranberry and lemon juice. The model predicted well the cell survival in orange, blackcurrant and pineapple, however it failed to predict cell survival in grapefruit and pomegranate, indicating the influence of additional factors, besides pH and citric acid, on cell survival. Very good cell survival (less than 0.4 log decrease) was observed after 6 weeks of storage in orange, blackcurrant and pineapple juice, all of which had a pH of about 3.8. Cell survival in cranberry and pomegranate decreased very quickly, whereas in the case of lemon juice, the cell concentration decreased approximately 1.1 logs after 6 weeks of storage, albeit the fact that lemon juice had the lowest pH (pH ~ 2.5) among all the juices tested. Taking into account the results from the compositional analysis of the juices and the model, it was deduced that in certain juices, other compounds seemed to protect the cells during storage; these were likely to be proteins and dietary fibre In contrast, in certain juices, such as pomegranate, cell survival was much lower than expected; this could be due to the presence of antimicrobial compounds, such as phenolic compounds.     

Extraction of resveratrol from the pomace of Palomino fino grapes by supercritical carbon dioxide

Resveratrol is a phenolic compound that is present in grapes and has significant benefits for human health. The development of methods to obtain concentrates of this compound is currently a major challenge in the food industry. In the work described here, resveratrol from grape seeds, stems, skin and pomace of the Palomino fino grape variety was extracted by supercritical carbon dioxide extraction. The effect of pressure (100, 400 bar), temperature (35, 55 °C) and the addition of modifier (5% v/v of ethanol) was evaluated to identify optimal resveratrol extraction from this by-product. Extraction yields and concentrations of resveratrol in the extracts were determined. The best results were obtained on working at high pressure and low temperature using 5% v/v ethanol as a co-solvent.     

Bacterial inactivation in fruit juices using a continuous flow Pulsed Light (PL) system

In this work, the susceptibility to pulsed light (PL) treatments of both a Gram-positive (L. innocua 11288) and a Gram-negative (E. coli DH5-??) bacteria inoculated in apple (pH = 3.49, absorption coefficient 13.9 cm^− 1) and orange juices (pH = 3.78, absorption coefficient 52.4 cm^− 1) was investigated in a range of energy dosages from 1.8 to 5.5 J/cm². A laboratory scale continuous flow PL system was set up for the experiments, using a xenon flash-lamp emitting high intensity light in the range of 100-1100 nm. The flashes lasted 360 ??s at a constant frequency of 3 Hz.The results highlighted how the lethal effect of pulsed light depended on the energy dose supplied, the absorption properties of liquid food as well as the bacterial strain examined. The higher the quantity of the energy delivered to the juice stream, the greater the inactivation level. However, the absorbance of the inoculated juice strongly influenced the dose deliver and, therefore, the efficiency of the PL treatment. Among the bacteria tested, E. coli cells showed a greater susceptibility to the PL treatment than L. innocua cells in both apple and orange juices. Following treatment at 4 J/cm², microbial reductions in apple and orange juices were, respectively, 4.00 and 2.90 Log-cycles for E. coli and 2.98 and 0.93 Log-cycles for L. innocua.Sublethally injured cells were also detected for both bacterial strains, thus confirming that membrane damage is an important event in bacterial inactivation by PL.     

Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log₁₀ cfu/well) and that they contained 1-2 log₁₀ cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log₁₀ cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log₁₀ more L. plantarum than L. monocytogenes cells (total count, 9 log₁₀ cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log₁₀ cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log₁₀ cfu/well inactivation after exposure for 15 min to 100 μg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log₁₀ cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log₁₀ cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms.     

Technological and safety properties of Lactobacillus plantarum strains isolated from a Tunisian traditional salted meat

A total of 17 strains of Lactobacillus plantarum, isolated from a Tunisian traditional salted meat and identified by biochemical and molecular methods, were characterized according to their technological properties including acidifying, antimicrobial and enzymatic activities as well as antibiotic resistance in order to select the most suitable for use as starter cultures for the production of fermented sausages. All the strains studied showed good acidifying activity and were able to reduce the pH to less than 4.3 in 72, 48 and 24h at 15, 25 and 37°C respectively. The majority of strains displayed antimicrobial activities against Salmonella arizonae, Staphylococcus aureus, Pseudomonas aeuroginosa and Escherichia coli, however characterization of the antimicrobial substances showed that none of the strains could produce bacteriocins. All the L. plantarum strains were able to hydrolyze casein, whereas none of them was found to possess lipolytic activity. The majority of strains of L. plantarum were resistant to tetracycline, erythromycin, rifampicin, ampicillin and penicillin G.     

Modeling the inactivation of Salmonella typhimurium by dense phase carbon dioxide in carrot juice

The inactivation of Salmonella typhimurium inoculated into acidified carrot juice subjected to dense phase carbon dioxide (DPCD) was investigated. The pressures in the study were 10, 20 and 30 MPa, the temperatures were 32, 37 and 42 °C, and the treatment time was 5–90 min. The inactivation effect of DPCD was enhanced by increasing pressure and temperature. The sigmoid inactivation curves were characterized with the lag phase, exponential inactivation phase, and resistant phase. The inactivation curves were fitted to the modified Gompertz equation and the modified Logistic equation, the modified Gompertz equation was superior since its lowest residual sum of squares (RSS) was lower although there was no significant difference of goodness-of-fit between both models as indicated by F-test. The λ (the duration of the lag phase) and t_4-D (the time necessary to achieve 4-log cycles reduction) decreased with increasing pressure or temperature. The k_dm (the maximum specific value of the inactivation rate, min⁻¹) increased with increasing temperatures, and decreased with increasing pressures. The activation energy (Ea) and the activation volume (Va) necessary for inactivating S. typhimurium by DPCD were 19.06–29.39 kJ mol⁻¹ and 18.89–58.27 cm³ mol⁻¹.     

Change of polyphenol oxidase activity, color, and browning degree during storage of cloudy apple juice treated by supercritical carbon dioxide

The effects of supercritical carbon dioxide (SCCO₂) treatment of 8, 15, 22, and 30 MPa for 60 min at 55 °C on polyphenol oxidase (PPO) activity, color, and browning degree in cloudy apple juice during storage at 4 °C for 4-weeks were investigated. The SCCO₂ treatment had significant effects on inactivation of PPO and the least residual activity of PPO was 38.50% at 30 MPa. The restoration of PPO residual activity after SCCO₂ treatment was also observed, which was dependent on the pressure level. A greater reduction of lightness L and a minor increase of redness a of cloudy apple juices after SCCO₂ treatment occurred. Moreover, the total color difference (ΔE), which was significantly less than that of untreated sample, was decreased by enhancing the pressure level. The changes of lightness L and browning degree A during storage were well fitted to a first-order kinetic model. The rate constants of k _L and k _A of samples subjected to SCCO₂ treatment reduced from 4.75×10⁻² to 0.42×10⁻² and 37.19×10⁻² to 8.02×10⁻², respectively, when pressure increased from 0 MPa (untreated sample) to 30 MPa.     

Inactivation and reactivation of horseradish peroxidase treated with supercritical carbon dioxide

Effects of supercritical carbon dioxide (SCCO₂) on the activity of horseradish peroxidase (HRP) in pH 5.6 acetate buffer solution were investigated. SCCO₂ treatment could effectively inactivate HRP. Higher pressure, higher temperature, and longer treatment time caused more inactivation. The maximum reduction of HRP activity reached nearly 90% at 30 MPa and 55 °C for 60 min. Analysis of first-order reaction kinetic data (characterized by a rate constant k and by a decimal reduction time D) showed that D value was closely related to the pressure and temperature of SCCO₂ treatment. Higher pressures or higher temperatures resulted in lower D values (higher k), the D value of HRP was minimized to 64.52 min treated by the combination of 30 MPa and 55 °C. The Z _p, representing the range of applied pressure between which the D values change by a factor of 10, was 114.81 MPa. The activity of HRP treated by SCCO₂ was reactivated significantly after initial 7-day storage at 4 °C apart from the samples at 30 MPa for 60 min, indicating the HRP inactivation may be reversible and the reactivation of HRP is dependent on the pressure level and treatment time.     

Application of response surface methodology to optimise supercritical carbon dioxide extraction of essential oil from Cyperus rotundus Linn.

Supercritical fluid extraction with carbon dioxide (SC-CO₂ extraction) was performed to isolate essential oils from the rhizomes of Cyperus rotundus Linn. Effects of temperature, pressure, extraction time, and CO₂ flow rate on the yield of essential oils were investigated by response surface methodology (RSM). The oil yield was represented by a second-order polynomial model using central composite rotatable design (CCRD). The oil yield increased significantly with pressure (p < 0.0001) and CO₂ flow rate (p < 0.01). The maximum oil yield from the response surface equation was predicted to be 1.82% using an extraction temperature of 37.6 °C, pressure of 294.4 bar, extraction time of 119.8 min, and CO₂ flow rate of 20.9 L/h.     

Inactivation of different strains of Escherichia coli O157:H7 in various apple ciders treated with dimethyl dicarbonate (DMDC) and sulfur dioxide (SO2) as an alternative method

Escherichia coli has been identified as the causative agent in numerous foodborne illness outbreaks associated with the consumption of fresh apple cider. Apple cider has a pH which is normally below 4.0 and would not be considered a medium capable of supporting the growth of foodborne pathogens. The association of unpasteurized apple cider with foodborne illness due to E. coli O157:H7 has however, led to increased interest in potential alternative methods to produce pathogen free cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10⁶–10⁷ CFU of three strains of E. coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895) and tested to determine the effectiveness of sulfur dioxide (SO₂) and dimethyl dicarbonate (DMDC). Bacterial populations for treated and untreated samples were then enumerated by using non-selective media. Eight different ciders were treated with DMDC (125 and 250 ppm) and SO₂ (25, 50, 75, 100 ppm). Greater than a 5-log reduction was achieved at room temperature with 250 ppm of DMDC and 50 ppm of SO₂ after the incubation time of 6 h and 24 h, respectively. Addition of DMDC and/or SO₂ may offer an inexpensive alternative to thermal pasteurization for the production of safe apple cider for small apple cider producers.     

Enzymatic synthesis of cocoa butter analog through interesterification of lard and tristearin in supercritical carbon dioxide by lipase

Substrate oil composition, reaction time, acyl donor, temperature, and pressure affected the triacylglycerol (TG) content of cocoa butter analog during the interesterification reaction catalyzed by lipase in a supercritical carbon dioxide (SC-CO₂) system. Among oil sources used to interact with tristearin, the content of 1(3)-palmitoyl-3(1)-stearoyl-2-monoolein (POS) (P, palmitate; O, oleate; S, stearate) and 1-palmitoyl-2, 3-dioleoylglycerol (POO) in analog was most similar to the corresponding TG content of cocoa butter when analog was prepared with lard. The optimized interesterification reaction using lard and tristearin (at a mole ratio of 1.4) as substrates to produce cocoa butter analog in a SC-CO₂ system was at 17 MPa, 50 °C, pH 9, for 3 h with an immobilized lipase, Lipozyme IM-20, from Mucor miehei. The lyophilized enzyme facilitated the production of cocoa butter analog in anhydrous substrates (a_w 0.33). The yield and melting point of the purified cocoa butter analog by a silica column was 63% and 34.5 °C, respectively, when the analog was produced under optimal conditions.     

Comparison of inactivation of Listeria monocytogenes within a biofilm matrix using chlorine dioxide gas,aqueous chlorine dioxide and sodium hypochlorite treatments

The present research compared the effect of chlorine dioxide (CD) gas, aqueous CD and aqueous sodium hypochlorite (SHC) treatments on the inactivation of a five strain mixture of Listeria monocytogenes – containing biofilms. Four day old biofilms were developed on a stainless steel (SS 304) coupon by using a mixture of five cultures of L. monocytogenes (Scott A, N1-227, 103M, 82 and 311) using a 100% relative humidity (RH) dessicator for incubation at room temperature (22 ± 2 °C). After biofilm development, coupons were rinsed and dried for 2 h and treated with 0.3 mg/l CD gas at 75% RH, 7 mg/l of aqueous CD and 50 mg/l SHC. Initial log₁₀ population of biofilm cells before CD gas, aqueous CD and SHC treatment was 4.80, 5.09 and 4.95 log₁₀ CFU/cm². The Weibull model was used to fit non-linear survivor curves. Treatments and time points of 0.3 mg/l CD gas and 7 mg/l aq. CD solution were significantly different (p < 0.05). A 10 min treatment of 0.3 mg/l CD gas, 7 mg/l of aq. CD, and 50 mg/l SHC resulted in reductions of 3.21, 3.74 and 3.09 log₁₀ CFU/cm², respectively. At 10 min, all treatments were not statistically different (p > 0.05). Low levels of CD (0.3 mg/l CD gas and 7 mg/l aq. CD solution) for 10 min resulted in similar log reductions compared to 50 mg/l SHC.