Characterization of bacteriocin bificin C6165: a novel bacteriocin

Aims

To purify and primarily characterize an anti‐Alicyclobacillus bacteriocin produced by Bifidobacterium animalis subsp. animalis CICC 6165, suggested to be named bificin C6165.

Methods and Results

During purification of the bificin C6165, optimal recovery was achieved with ammonium sulfate precipitation followed by two chromatographic steps. Mass spectrometry analyses revealed a distinctive peak corresponding to a molecular mass of 3395·1 Da. This bacteriocin was heat stable, effective after refrigerated storage and freeze–thaw cycles. The primary mode of action of bificin C6165 is most probably due to pore formation, as indicated by the efflux of K⁺ from metabolically active cells of Alicyclobacillus acidoterrestris. In the presence of 10 mmol l^?1 gadolinium, bificin C6165 did not affect cells of Alicyclobacillus acidoterrestris. This suggests that the mode of action of bificin C6165 relies on a net negatively charged cell surface.

Conclusions

Bificin C6165 is indeed a novel bacteriocin and it exhibited remarkable potency for Alicyclobacillus control.

Significance and Impact of the Study

Application of bacteriocins in preservation of fruit juices has seldom been studied. Bificin C6165 may be an alternative method to control juice spoilage by this Alicyclobacillus acidoterrestris and meet increasing consumer demand for nature and artificial chemical additive‐free food products.     

Temperature, soluble solids and pH effect on Alicyclobacillus acidoterrestris viability in lemon juice concentrate

Alicyclobacillus acidoterrestris is a thermoacidophilic, non-pathogenic, spore-forming bacterium detected in spoiled commercial pasteurized fruit juice. Apple, white grape and tomato are particularly susceptible. A. acidoterrestris spores are resistant to lemon juice pasteurization (2 min at 82°C), and they can germinate and grow causing spoilage. This contamination is characterized by a medicinal or disinfectant smell attributed to guaiacol (o-dihydroxybenzene) production and other taint chemicals. The aim of this work was to study the influence of temperature (82, 86, 92 and 95 °C), total soluble solids (SS) (6.20, 9.8, 50 and 68°Brix) and pH (2.28, 2.45, 2.80, 3.25, 3.5) on decimal reduction time (D) of the A. acidoterrestris in clarified and non-clarified concentrated lemon juice. Once D-value was determined, the resistance of A. acidoterrestris at the assayed temperatures was confirmed. SS and pH influence spore viability, because spore resistance increases with higher SS (50°Brix 22 min 82 °C–68°Brix 28 min 82 °C) and pH values (pH 2.28, 17 min–pH 4.00, 22 min). Bacterial growth was lower in clarified lemon juice, 26 min at 82 °C, than in non-clarified lemon juice, 51 min at 82 °C. Temperature was the parameter that had the greatest influence on the D value.     

Influence of Different Filling,Cooling, and Storage Conditions on the Growth of Alicyclobacillus acidoterrestris CRA7152 in Orange Juice

The prevention of spoilage by Alicyclobacillus acidoterrestris is a current challenge for fruit juice and beverage industries worldwide due to the bacterium''s acidothermophilic growth capability, heat resistance, and spoilage potential. This study examined the effect of storage temperature on A. acidoterrestris growth in hot-filled orange juice. The evolution of the A. acidoterrestris population was monitored under six different storage conditions after pasteurization (at 92°C for 10 s), maintenance at 85°C for 150 s, and cooling with water spray to 35°C in about 30 min and using two inoculum levels: <10¹ and 10¹ spores/ml. Final cooling and storage conditions were as follows: treatment 1, 30°C for the bottle cold point and storage at 35°C; treatment 2, 30°C for 48 h and storage at 35°C; treatment 3, 25°C for the bottle cold point and storage at 35°C; treatment 4, 25°C for 48 h and storage at 35°C; treatment 5, storage at 20°C (control); and treatment 6, filling and storage at 25°C. It was found that only in treatment 5 did the population remain inhibited during the 6 months of orange juice shelf life. By examining treatments 1 to 4, it was observed that A. acidoterrestris predicted growth parameters were significantly influenced (P < 0.05) either by inoculum level or cooling and storage conditions. The time required to reach a 10⁴ CFU/ml population of A. acidoterrestris was considered to be an adequate parameter to indicate orange juice spoilage by A. acidoterrestris. Therefore, hot-filled orange juice should be stored at or below 20°C to avoid spoilage by this microorganism. This procedure can be considered a safe and inexpensive alternative to other treatments proposed earlier.The first Alicyclobacillus sp. discovered was isolated in 1982 from spoiled apple juice aseptically packed in Germany and was considered at that time strictly limited to thermophilic and acidic environments (5). The spoilage of fruit juices by Alicyclobacillus is characterized by “off” flavors (medicinal or phenolic) due to guaiacol, 2,6-dibromophenol, and 2,6-dichlorophenol (20, 32, 25). As the spoilage does not show any evident signs like swelling of the container or any overt changes in the fruit juice (e.g., pH or turbidity), it is often not recognized until the packages are opened, the product is tasted, and consumer complaints are received by the manufacturer (6).Alicyclobacillus acidoterrestris, Alicyclobacillus cycloheptanicus and Alicyclobacillus acidocaldarius were the first three species described when the Alicyclobacillus genus was created in 1992 (31). Although there are currently more than 15 species described (27), only four Alicyclobacillus species (A. acidoterrestris, A. pomorum, A. herbarius and A. acidophilus) have shown the ability to produce off flavors in fruit juices or beverages (5, 13, 9, 1). Of these, A. acidoterrestris is considered the most important spoilage species within the Alicyclobacillus genus either by its frequency of occurrence or by its linkage to the spoilage problems of fruit juices and beverages. The broad temperature range for A. acidoterrestris growth (25 to 60°C) (33, 21, 20, 30, 11), its ability to grow under acidic environments (pH 2.5 to 6.0) (20, 30, 19, 22, 6), and its high heat resistance in orange juice (D at 95°C of 2.7 min) (8) together provide adequate conditions for both survival through pasteurization and growth during juice storage.A. acidoterrestris growth and the consequent orange juice spoilage can lead to enormous economic losses; therefore, this microorganism is currently considered a major challenge for the fruit juice industries. It is known to be difficult, if not impossible, to guarantee the absence of Alicyclobacillus spores on the surface of fruits used to make juices since the soil is the primary niche of Alicyclobacillus spp. (7). Subsequently, control measures such as avoiding fruit contact with soil and the use of sanitizers during the fruit washing step before crushing have been studied (16, 12). In addition, fruit juice producers need to better control the pasteurization conditions and to redesign their thermal processes for targeting Alicyclobacillus spp. (28, 24). However, the limited effectiveness of sanitizers against Alicyclobacillus spores and the sensory and nutritional problems that may arise from increased time and temperature regimes in pasteurization are recognized. Since the complete inactivation of Alicyclobacillus spores from raw materials may not be feasible and since juice spoilage by this microorganism depends on the germination of spores and outgrowth, studies reporting conditions that avoid spoilage by controlling Alicyclobacillus spore germination are necessary. The best option to manage the challenge that Alicyclobacillus presents to the fruit juice industries will be one that results in the fewest alterations in processing and storage conditions, preserves the nutritional and sensory aspects of the final products, does not impact production costs and commercial practices, and at the same time ensures the control of the microorganism.In the fruit juice industry, two main types of thermal processes are commonly applied: pasteurization followed by a hot-fill process or pasteurization followed by a cold-fill process (6). In the former, after the product is heated to >90 to 95°C, it is held hot for 15 to 20 s. As the temperature decreases to 82 to 84°C, the product is filled into the package. Next, the product is held for approximately 2 min before the packages are cooled to room temperature. Hot filling has been extensively used in the manufacturing processes of fruit-based drinks and beverages, but problems due to spoilage caused by Alicyclobacillus may arise during fruit juice shelf life. This is due to the extended time that the product is maintained at temperature conditions adequate for the germination and outgrowth of acidothermophilic spore-forming microbes. Despite several studies regarding the factors that affect A. acidoterrestris growth and heat resistance (12, 18, 2, 29), there is a lack of research on the effects of hot-filled fruit juice storage conditions on A. acidoterrestris growth during juice storage. Thus, this study aimed at estimating and comparing the growth parameters (maximum population ratio, κ; lag time, λ; and maximum growth rate, μ) of A. acidoterrestris survival in hot-filled orange juice that was cooled and stored under several conditions that simulate industrial and commercial practices. Primary growth parameters were estimated by using the Baranyi predictive model (3). Additionally, orange juice cooling and storage conditions that avoided germination, growth, and guaiacol production by A. acidoterrestris CRA 7152 were determined.     

Purification and Characterization of Warnericin RB4, Anti-Alicyclobacillus Bacteriocin, Produced by Staphylococcus warneri RB4

In this study we characterized a bacteriocin, warnericin RB4, produced by Staphylococcus warneri RB4. Warnericin RB4 activity was completely inactivated by trypsin and actinase E. The activity was stable at 100°C for 15 min, and had a pH range of 2 to 6. S. warneri RB4 showed antibacterial activity against only Alicyclobacillus acidoterrestris, A. acidocaldarius, and Micrococcus luteus, among 34 bacterial species tested. The amino acid sequence of the purified bacteriocin contained 27 amino acid residues (K-K-K-S-G-V-I-P-X-V-X-H-D-X-H-M-N-X-F-Q-F-V-F-X-X-X-S). The molecular mass of the bacteriocin was estimated to be 2,958.2 Da by ESI-MS. These results show that the Warnericin RB4 exhibiting specific antibacterial activity against thermo-acidophiles, Alicyclobacillus spp., is a Nukacin ISK-1 or closely related bacteriocin, classified with class IA (Lacticin 481 types). This is the first report that Warnericin RB-4 is effective to inhibit the growth of causative microorganisms of spoilage in various acidic drinks. Warnericin RB4 might prove useful in fruit juices and fruit juice–containing drinks.     

Immobilization of tomato (Lycopersicon esculentum) pectinmethylesterase in calcium alginate beads and its application in fruit juice clarification

Clarity of fruit juices is desirable to maintain an aesthetically pleasing quality and international standards. The most commonly used enzymes in juice industries are pectinases. A partially-purified pectinmethylesterase from tomato was entrapped in calcium alginate beads and used for juice clarification. The activity yield was maximum at 1 % (w/v) CaCl₂ and 2.5 % (w/v) alginate. The immobilized enzyme retained ~55 % of its initial activity (5.7 × 10^?2 units) after more than ten successive batch reactions. The K_m, pH and temperature optima were increased after immobilization. The most effective clarification of fruit juice (%T₆₂₀ ~60 %) by the immobilized enzyme was at 4 °C with a holding time of 20 min. The viscosity dropped by 56 % and the filterability increased by 260 %. The juice remains clear after 2 months of storage at 4 °C.     

Inactivation of Alicyclobacillus acidoterrestris vegetative cells in model system,apple, orange and tomato juices by high hydrostatic pressure

The objective of this study is to determine the effect of high hydrostatic pressure (HHP) on inactivation of Alicyclobacillus acidoterrestris vegetative cells in a model system (BAM broth) and in orange, apple and tomato juices. The shelf-life stability of pressurized juices is also studied. In general the viability loss was enhanced significantly as the level of pressure and temperature were increased (P < 0.05). 4.70 log cycle reduction was obtained after pressurization at 350 MPa at 50 °C for 20 min in BAM broth whereas thermal treatment at 50 °C for 20 min caused only 1.13 log cycle inactivation showing the effectiveness of HHP treatment on inactivation. The D values for pressure (350 MPa at 50 °C) and temperature (50 °C) treatments were 4.37 and 18.86 min in BAM broth, respectively. All juices were inoculated with A. acidoterrestris cells to 10⁶ c.f.u./ml and were pressurized at 350 MPa at 50 °C for 20 min. More than 4 log cycle reduction was achieved in all juices studied immediately after pressurization. The pressurized juices were also stored up to 3 weeks at 30 °C and the viable cell numbers of A. acidoterrestris in orange, apple and tomato juices were 3.79, 2.59 and 2.27 log cycles, respectively after 3 weeks. This study has indicated that A. acidoterrestris vegetative cells can be killed by HHP at a predictable rate even at temperatures at which the microorganism would normally grow.     

Quorum-Sensing Regulation of Constitutive Plantaricin by Lactobacillus plantarum Strains under a Model System for Vegetables and Fruits

This study aimed at investigating the regulatory system of bacteriocin synthesis by Lactobacillus plantarum strains in vegetables and fruits in a model system. Sterile and neutralized cell-free supernatant (CFS) from L. plantarum strains grown in MRS broth showed in vitro antimicrobial activities toward various indicator strains. The highest activity was that of L. plantarum C2. The antimicrobial activity was further assayed on vegetable and fruit agar plates (solid conditions) and in juices (liquid conditions). A regulatory mechanism of bacteriocin synthesis via quorum sensing was hypothesized. The synthesis of antimicrobial compounds seemed to be constitutive under solid conditions of growth on vegetable and fruit agar plates. In contrast, it depended on the size of the inoculum when L. plantarum C2 was grown in carrot juice. Only the inoculum of ca. 9.0 log CFU ml⁻¹ produced detectable activity. The genes plnA, plnEF, plnG, and plnH were found in all L. plantarum strains. The genes plnJK and plnN were detected in only three or four strains. Reverse-phase high-performance liquid chromatography purification and mass spectrometry analysis revealed the presence of a mixture of eight peptides in the most active fraction of the CFS from L. plantarum C2. Active peptides were encrypted into bacteriocin precursors, such as plantaricins PlnJ/K and PlnH and PlnG, which are involved in the ABC transport system. A real-time PCR assay showed an increase in the expression of plnJK and plnG during growth of L. plantarum C2 in carrot juice.     

Fate ofescherichia coli andE. coli O157∶H7 in apple juice treated with propolis extract

Fruit juices are targets of spoilage moulds, yeasts and acid tolerant bacteria. They might be contaminated with bacteria from raw materials, environment, packaging and during the handling of the product. These contaminations have frequently resulted in the spoilage of fruit juice and consequently commercial losses. The objective of this study was to determine the influence of propolis in apple juice againstEscherichia coli andE. coli O157:H7 strains of the spoilage and pathogenic bacteria. For this purpose, apple juice was obtained from fresh apples and then was pasteurised. The pH value, titrable acidity (as % malic acid) and Brix degree of this apple juice were 3.72±0.10, 0.67±0.05% and 12.1±0.01, respectively. Propolis extract at 1,2 and 5% concentrations were tested to determine ofE. coli andE. coli O157:H7 inhibition using paper disc diffusion method. The control treatment had no propolis extract. The apple juices were contaminated with these bacteria, and the activity of propolis was observed at first, 18^th, 24^th, 48^th and 72^nd hours at 4 and 25°C. The number of cells in the tubes was counted using serial dilution method. Results indicated that propolis extract at 2 and 5% concentrations had significant antimicrobial activity againstE. coli andE. coli O157:H7, therefore we can conclude that propolis extract is worthy of further study as a natural preservative for the foods prone to microbial spoilage.     

Potential of patulin production by Penicillium expansum strains on various fruits

In this study, we investigated the pathogenicity and patulin production by ten strains of Penicillium expansum on various fruits (apples, apricots, kiwis, plums and peaches) at two (4°C and 25°C) different temperature regimes. All strains caused the infectious rots on all fruits at 4 and 25°C except one strain (PEX 09) at 4°C. Two strains (PEX 20 and PEX 12) out of ten produced the highest amounts of patulin on all fruits tested. The patulin production by P. expansum is high at 25°C compared to 4°C. All strains of P. expansum accumulated patulin ranging from 100–13,200 μg/kg and nine strains ranging from 100–12,100 μg/kg in all fruits at 25°C and 4°C, respectively. Among ten strains of P. expansum, strain PEX 20 produced the greatest amount of patulin on apricots (13,200 μg/kg of rotten fruit) and on apples (12,500 μg/kg) at 25°C after 9 days of incubation. At 4°C, this strain produced 12,100, 12,000, 2,100 and 1,200 μg/kg of patulin on apricots, apples, plums and peaches, respectively, after 45 days of incubation. Strain PEX 12 produced the highest amount of patulin on kiwis (10,700 μg/kg) at 25°C and 10,300 μg/kg at 4°C. Patulin production by P. expansum on peaches and plums at both temperatures were lower than other fruits. The results of this study showed that careful removal of rotten fruits is essential to produce patulin-free fruit juice, since high patulin levels in apricots, apples and kiwis could result in a level greater than 50 μg/kg of this mycotoxin in finished fruit juices, when one contaminated fruit occurs in 264, 250 and 214 fruits, respectively. So, the fruit processors should take care in not using rotten fruits for juice production to avoid the patulin problem worldwide, since this study proved that most important fruits being used for juice production and direct human consumption are susceptible to P. expansum and subsequent patulin production even at low temperatures. This is the first comprehensive report regarding patulin production by different strains of P. expansum on various fruits from Italy at different temperature regimes.     

Characterization of a bacteriocin produced by Lactococcus lactis subsp. lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery

Lactococcus lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery inhibits the growth of Citrobacter freundii (a bullfrog pathogen) and Listeria monocytogenes by a synergistic effect between lactic acid, hydrogen peroxide and a bacteriocin-like molecule. The chemical characterization of the bacteriocin in cell-free supernatants indicates that it has a proteinaceous nature. Hexadecane and ethyl acetate did not modify the bacteriocin activity, while 10 and 20 % (v/v) chloroform decreased the activity by 29 and 43 %, respectively. The antimicrobial peptide was heat stable since 85 % of residual activity was detected when neutralized supernatants were heated at 80 °C for 30 min. Moreover, no bacteriocin inactivation was observed when supernatants were kept at ?20 °C for 3 months. The synthesis of the bacteriocin was associated with bacterial growth, highest production (2,100 AU/ml) being detected at the end of the exponential growth phase. At pH ranges of 5–6.5 and 5.0–5.5 the inhibitory molecule was stable when stored for 2 days at 4 and 25 °C, respectively. Moreover, it had a bactericidal effect on L. monocytogenes and the ultrastructural studies of pathogenic cells revealed clumping of the cytoplasmic material, increased periplasmic space and cell wall modifications. The deduced amino acid sequence of the bacteriocin was identical to nisin Z and the genetic determinants for its production are harbored in the chromosome. These results, described for the first time in L. lactis from a bullfrog hatchery, will increase knowledge of the bacteriocin under study with a view to its potential inclusion in probiotics for raniculture or biopreservatives.     

Inhibitory Effects of High Pressure and Heat on Alicyclobacillus acidoterrestris Spores in Apple Juice

The effectiveness of combined high pressure and heat treatment for reducing spore levels of Alicyclobacillus acidoterrestris, a thermoacidophilic spore-forming bacterium, in commercial pasteurized apple juice was investigated. Spores suspended in apple juice were successfully destroyed by combining high pressure with a mild or high temperature (45, 71, or 90°C).     

Purification and Characterization of Bacteriocin Produced by Lactobacillus brevis UN Isolated from Dhulliachar: a Traditional Food Product of North East India

A bacteriocin producing strain Lactobacillus brevis UN isolated from Dulliachar—a salted pickle and identified by biochemical and molecular methods. L. brevis UN was found to produce bacteriocin with broad spectrum activity against spoilage causing/food borne pathogens viz. L. monocytogenes, C. perfringens, S. aureus, L. mesenteroides, L. plantarum and B. cereus. Bacteriocin production was optimized through classical one variable at a time method. The isolate showed maximum bacteriocin production at early stationary phase, pH 4.0, temperature 35 °C and with an inoculum size of 1.5 OD @ 10 %. Bacteriocin produced by L. brevis UN was purified to homogeneity by single step gel exclusion chromatography and was most active at pH 6.0 and 7.0, stable up to 100 °C and was proteinaceous in nature. The results of NMR revealed the presence of proline, glutamic acid, aspartic acid, leucine, isoleucine and serine in its peptide structure. PCR amplification analysis determined that bacteriocin encoded gene in L. brevis UN was plasmid bound.     

Genotypic and Phenotypic Heterogeneity in Alicyclobacillus acidoterrestris: A Contribution to Species Characterization

Alicyclobacillus acidoterrestris is the main cause of most spoilage problems in fruit juices and acidic products. Since soil borne species often contaminate fruit juices and do not need strict extreme requirements for survival, it is a great concern to investigate whether and how soil species could evolve from their ecological niches in microbial community to new environments as fruit juices. In this study, 23 isolates of thermo-acidophilic, spore-forming bacteria from soil were characterized by cultural and molecular methods. In addition, 2 strains isolated from a spoilage incident in pear juice were typed. Strains phenotyping showed that they could be grouped into 3 different clusters, and some isolates showed identical or quite similar patterns. Analyzing pH and temperature ranges for growth, the majority of strains were able to grow at values described for many species of Alicyclobacillus. Qualitative utilization of lysine, arginine and indole production from tryptophan revealed, for the first time, deamination of lysine and decarboxylation of arginine. Resistance to 5% NaCl as well as the ability to hydrolyze starch and gelatin, nitrate reduction, catalase and oxidase activities confirmed literature evidences. Examining of 16S rRNA, showed that isolates were divided into three blocks represented by effectively soil species and strains that are moving from soil to other possible growing source characterized by parameters that could strongly influence bacterial survival. RAPD PCR technique evidenced a great variability in banding patterns and, although it was not possible to obtain genotypically well-distinguished groups, it was feasible to appreciate genetic similarity between some strains. In conclusion, the investigation of a microbial community entails a combination of metagenomic and classic culture-dependent approaches to expand our knowledge about Alicyclobacillus and to look for new subspecies.     

Immunomagnetic Separation Combined with Polymerase Chain Reaction for the Detection of Alicyclobacillus acidoterrestris in Apple Juice

A combination of immunomagnetic separation (IMS) and polymerase chain reaction (PCR) was used to detect Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice. The optimum technological parameters of the IMS system were investigated. The results indicated that the immunocapture reactions could be finished in 60 min and the quantity of IMPs used for IMS was 2.5 mg/mL. Then the combined IMS-PCR procedure was assessed by detecting A. acidoterrestris in apple juice samples. The agarose gel electrophoresis results of 20 different strains showed that the IMS-PCR procedure presented high specificity to the A. acidoterrestris. The sensitivity of the IMS-PCR was 2×10¹ CFU/mL and the total detection time was 3 to 4 h. Of the 78 naturally contaminated apple juice samples examined, the sensitivity, specificity and accuracy of IMS-PCR compared with the standardized pour plate method were 90.9%, 97.0% and 96.2%, respectively. The results exhibited that the developed IMS-PCR method will be a valuable tool for detecting A. acidoterrestris and improving food quality in juice samples.     

Bacteriocin-Producing Lactic Acid Bacteria Isolated from Mangrove Forests in Southern Thailand as Potential Bio-Control Agents: Purification and Characterization of Bacteriocin Produced by Lactococcus lactis subsp. lactis KT2W2L

The aim of this work was to purify and characterize the bacteriocin produced by Lactococcus lactis subsp. lactis KT2W2L previously isolated from mangrove forests in southern Thailand, in order to evaluate its potential as new food protective agent. The active peptide from the cell-free supernatant of this strain was purified in 4 steps: (1) precipitation with 70 % saturated ammonium sulfate, (2) elution on a reversed-phase cartridge using different concentrations of acetonitrile, (3) cation-exchange chromatography and (4) final purification by reversed-phase HPLC on a C₈ column. The molecular mass of 3,329.5254 Da of the purified bacteriocin, determined by mass spectrometry, is nearly identical to that of peptide nisin Z. The activity of the purified bacteriocin was unaffected by pH (2.0–10.0), thermostable but was sensitive to proteolytic enzymes. The bacteriocin activity was stable after 8 weeks of storage at ?20 °C and 7 weeks of storage at 4 °C, but decreased after 3 weeks of storage at 37 °C. It was stable when incubated for 1 month at 4 °C in 0–30 % NaCl. Inhibitory spectrum of this bacteriocin showed a wide range of activity against similar bacterial strains, food-spoilage and food-borne pathogens. L. lactis subsp. lactis KT2W2L was sensitive to kanamycin, penicillin and tetracycline but resistant to ampicillin, gentamicin and vancomycin. The fragment obtained after amplification of genomic DNA from L. lactis subsp. lactis KT2W2L, with specific primers for bacteriocin genes, presented 99 % homology to the nisin Z gene. PCR amplification demonstrated that L. lactis subsp. lactis KT2W2L does not harbor virulence genes cylA, cylB, efaAfs and esp. The bacteriocin and its producing strain may find application as bio-preservatives for reduction in food-spoilage and food-borne pathogens in food products.     

Assessment of synergistic combination potential of probiotic and bacteriophage against antibiotic-resistant Staphylococcus aureus exposed to simulated intestinal conditions

This study was designed to evaluate the combined effect of probiotic Lactobacillus rhamnosus and bacteriophage SA11 on the control of antibiotic-sensitive Staphylococcus aureus (ASSA) and antibiotic-resistant S. aureus (ARSA) under the simulated intestinal conditions. The survivability of ASSA and ARSA were determined in the simulated phosphate-buffered saline (PBS)-, trypticase soy broth (TSB)-, and milk-based gastric juices adjusted to pH 2.0, 3.0, and 5.0 at 37 °C for 30 min. The inhibitory effect of bacteriophage SA11 and probiotic on the growth of ASSA and ARSA was evaluated in the simulated intestinal juices at 37 °C for 20 h. The least reductions in the numbers of ASSA and ARSA were observed in the milk-based gastric juices at pH 2.0 (<1 log). No significant changes in the teichoic acid-mediated sliding motility were observed for ASSA and ARSA after 30-min exposure to the simulated gastric juices (pH 2.0, 3.0, and 5.0), responsible for the enhanced bacterial attachment to the epithelial cells. The bacteriophage SA11 was stable down to pH 5.0 and up to 0.06 % bile salts. The bacteriophage SA11 combined with probiotic effectively inhibited the growth of ASSA and ARSA in the simulated intestinal conditions, showing more than 4 log reduction. The relative expression levels of adhesion-related genes (clfA, eno, and fnbA) and efflux-related genes (mdeA, norB, and norC) were less decreased in ARSA than in ASSA after exposure to the simulated gastrointestinal conditions. These results might shed light on the application of bacteriophage to control the ingested antibiotic-resistant foodborne pathogens in the intestinal tract.     

Purification and antibacterial mechanism of fish-borne bacteriocin and its application in shrimp (<Emphasis Type="Italic">Penaeus vannamei</Emphasis>) for inhibiting <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Vibrio parahaemolyticus: is recognized as the main cause of gastroenteritis associated with consumption of seafood. Bacteriocin-producing Lactobacillus plantarum FGC-12 isolated from golden carp intestine had strong antibacterial activity toward V. parahaemolyticus. The fish-borne bacteriocin was purified by a three-step procedure consisting of ethyl acetate extraction, gel filtration chromatography and high performance liquid chromatography. Its molecular weight was estimated at 4.1 kDa using SDS-PAGE. The fish-borne bacteriocin reached the maximum production at stationary phase after 20 h. It was heat-stable (30 min at 121?°C) and remained active at pH range from 3.0 to 5.5, but was sensitive to nutrasin, papain and pepsin. Its minimum inhibitory concentration for V. parahaemolyticus was 6.0 mg/ml. Scanning electron microscopy analysis showed that the fish-borne bacteriocin disrupted cell wall of V. parahaemolyticus. The antibacterial mechanism of the fish-borne bacteriocin against V. parahaemolyticus might be described as action on membrane integrity in terms of the leakage of electrolytes, the losses of Na⁺K⁺-ATPase, AKP and proteins. The addition of the fish-borne bacteriocin to shrimps leaded V. parahaemolyticus to reduce 1.3 log units at 4?°C storage for 6 day. Moreover, a marked decline in total volatile base nitrogen and total viable counts was observed in bacteriocin treated samples than the control. It is clear that this fish-borne bacteriocin has promising potential as biopreservation for the control of V. parahaemolyticus in aquatic products.     

Bioconversion of Agave tequilana fructans by exo-inulinases from indigenous Aspergillus niger CH-A-2010 enhances ethanol production from raw Agave tequilana juice

Agave tequilana fructans are the source of fermentable sugars for the production of tequila. Fructans are processed by acid hydrolysis or by cooking in ovens at high temperature. Enzymatic hydrolysis is considered an alternative for the bioconversion of fructans. We previously described the isolation of Aspergillus niger CH-A-2010, an indigenous strain that produces extracellular inulinases. Here we evaluated the potential application of A. niger CH-A-2010 inulinases for the bioconversion of A. tequilana fructans, and its impact on the production of ethanol. Inulinases were analyzed by Western blotting and thin layer chromatography. Optimal pH and temperature conditions for inulinase activity were determined. The efficiency of A. niger CH-A-2010 inulinases was compared with commercial enzymes and with acid hydrolysis. The hydrolysates obtained were subsequently fermented by Saccharomyces cerevisiae to determine the efficiency of ethanol production. Results indicate that A. niger CH-A-2010 predominantly produces an exo-inulinase activity. Optimal inulinase activity occurred at pH 5.0 and 50 °C. Hydrolysis of raw agave juice by CH-A-2010 inulinases yielded 33.5 g/l reducing sugars, compared with 27.3 g/l by Fructozyme^® (Novozymes Corp, Bagsværd, Denmark) and 29.4 g/l by acid hydrolysis. After fermentation of hydrolysates, we observed that the conversion efficiency of sugars into ethanol was 97.5 % of the theoretical ethanol yield for enzymatically degraded agave juice, compared to 83.8 % for acid-hydrolyzed juice. These observations indicate that fructans from raw Agave tequilana juice can be efficiently hydrolyzed by using A. niger CH-A-2010 inulinases, and that this procedure impacts positively on the production of ethanol.     

Pseudomonas putida Strain FStm2 Isolated from Shark Skin: A Potential Source of Bacteriocin

Bacteriocin-producing Pseudomonas putida strain FStm2 isolated from shark showed broad range of antibacterial activity against all pathogens tested except Bacillus subtilis ATCC11774, MRSA N32064, Proteus mirabilis ATCC12453, Enterococcus faecalis ATCC14506, Salmonella typhimurium ATCC51312, Salmonella mutan ATCC25175, and Aeromonas hydrophila Wbf314. Of the three growth media tested in this study, TSB was observed to support the bacteriocin activity the most. While the highest bacteriocin activity was observed for media supplemented with 1 % NaCl, there was an observed reduction in bacteriocin activity with increasing salt concentration. Although the least bacteriocin activity was observed for marine broth, addition of increasing amounts of tryptone, glucose, or yeast extract increased bacteriocin activity. This was, however, contrary to the effect observed when MgSO₄ and MnSO₄ were added as supplements. In the presence of α-amylase, lipase, DNase, and RNase, a positive effect on bacteriocin production was observed. Proteinase K strongly inhibited bacteriocin production. Furthermore, the bacteriocins produced were heat stable within the temperature range of 30–70 °C. Bacteriocin activity also was not affected within a wide pH range of 3–9. Exposure to detergents did not inhibit the activity of the bacteriocin at the concentrations tested. Instead, a positive effect on the relative activity of produced bacteriocin was observed as sodium dodecyl sulfate (SDS), EDTA, and Tween 20 at 1 % concentration all improved bacteriocin activity when the cell-free supernatant was tested against Serratia marcescens ATCC 13880. The bacteriocin was purified by ammonium sulfate precipitation and gel filtration on a Superdex-200 column. SDS-PAGE analysis of the partially purified bacteriocin revealed an apparent molecular weight of ~32 kDa.     

Evaluation of different growth media for the recovery of the species of Alicyclobacillus

AIMS: Five different isolation media, namely potato dextrose agar (PDA), orange serum agar (OSA), K agar, yeast-starch-glucose agar and Bacillus acidocaldarius medium were evaluated for the recovery of Alicyclobacillus spp. from inoculated diluted and undiluted fruit-juice concentrates. METHODS AND RESULTS: Plates of PDA (pH 3.7), spread with vegetative cells (3.9 x 10(6) CFU ml(-1)) of Alicyclobacillus acidoterrestris from single-strength pear juice, recovered 2.9 x 10(6 )CFU ml(-1) after 5 days at 50 degrees C (74% recovery). The recovery of endospores from single-strength pear juice, after a heat treatment at 80 degrees C for 10 min, was higher on spread plates of OSA (pH 5.5) at 50 degrees C for 5 days (97% recovery). CONCLUSIONS: PDA (pH 3.7) and OSA (pH 5.5) at 50 degrees C for 3-5 days recovered the highest numbers of vegative cells and endospores of Alicyclobacillus spp. from sterilized fruit juices and concentrates. SIGNIFICANCE AND IMPACT OF THE STUDY: The most appropriate synthetic media for the recovery of Alicyclobacillus species from inoculated fruit juices and concentrates are shown.