Detection and activity of a bacteriocin produced by Leuconostoc mesenteroides.

Leuconostoc mesenteroides UL5 was found to produce a bacteriocin, referred as mesenterocin 5, active against Listeria monocytogenes strains but with no effect on several useful lactic acid bacteria. The antimicrobial substance is a protein, since its activity was completely destroyed following protease (pronase) treatment. However, it was relatively heat stable (100 degrees C for 30 min) and partially denaturated by chloroform. The inhibitory effect of the bacteriocin on sensitive bacterial strains was determined by a critical-dilution micromethod. Mutants of L. mesenteroides UL5 which had lost the capacity to produce the bacteriocin were obtained. The mutant strain was stable and phenotypically identical to parental cells and remained resistant to the bacteriocin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect bacteriocin activity corresponding to an apparent molecular mass of about 4.5 kDa.     

Detection and activity of a bacteriocin produced by Leuconostoc mesenteroides.

Leuconostoc mesenteroides UL5 was found to produce a bacteriocin, referred as mesenterocin 5, active against Listeria monocytogenes strains but with no effect on several useful lactic acid bacteria. The antimicrobial substance is a protein, since its activity was completely destroyed following protease (pronase) treatment. However, it was relatively heat stable (100 degrees C for 30 min) and partially denaturated by chloroform. The inhibitory effect of the bacteriocin on sensitive bacterial strains was determined by a critical-dilution micromethod. Mutants of L. mesenteroides UL5 which had lost the capacity to produce the bacteriocin were obtained. The mutant strain was stable and phenotypically identical to parental cells and remained resistant to the bacteriocin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect bacteriocin activity corresponding to an apparent molecular mass of about 4.5 kDa.     

Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus

A total of 52 strains of Lactobacillus acidophilus were examined for production of bacteriocins. A majority (63%) demonstrated inhibitory activity against all members of a four-species grouping of Lactobacillus leichmannii, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus lactis. Four L. acidophilus strains with this activity also inhibited Streptococcus faecalis and Lactobacillus fermentum, suggesting a second system of antagonism. Under conditions eliminating the effects of organic acids and hydrogen peroxide, no inhibition of other gram-positive or -negative genera was demonstrated by L. acidophilus. The agent produced by L. acidophilus N2 and responsible for inhibition of L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis was investigated. Ultrafiltration studies indicated a molecular weight of approximately 100,000 for the crude inhibitor. The agent was sensitive to proteolytic enzymes and retained full activity after 60 min at 100 degrees C (pH 5). Activity against sensitive cells was bactericidal but not bacteriolytic. These characteristics identified the inhibitory agent as a bacteriocin, designated lactacin B. Examination of strains of L. acidophilus within the six homology groupings of Johnson et al. (Int. J. Syst. Bacteriol. 30:53-68, 1980) demonstrated that production of the bacteriocin lactacin B could not be used in classification of neotype L. acidophilus strains. However, the usefulness of employing sensitivity to lactacin B in classification of dairy lactobacilli is suggested.     

Purification and partial characterization of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088

Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.     

Purification and partial characterization of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088.

Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.     

Partial purification and characterization of bacteriocin produced by Enterococcus faecalis DU10 and its probiotic attributes

A novel bacteriocin produced by avian duck isolated lactic acid bacterium Enterococcus faecalis DU10 was isolated. This bacteriocin showed a broad spectrum of antibacterial activity against important food-borne pathogens and was purified by size exclusion chromatography followed by reverse-phase high-performance liquid chromatography in a C-18 column. Tricine–SDS PAGE revealed the presence of a band with an estimated molecular mass of 6.3?kDa. The zymogram clearly linked the antimicrobial activity with this band. This result was further confirmed by mass-assisted laser desorption ionization time-of-flight mass spectrometry, since a sharp peak corresponding to 6.313?kDa was detected and the functional groups were revealed by Fourier transform infrared spectroscopy. Bacteriocin DU10 activity was found sensitive to proteinase-K and pepsin and partially affected by trypsin and α-chymotrypsin. The activity of bacteriocin DU10 was partially resistant to heat treatments ranging from 30 to 90°C for 30?min. It also withstood a treatment at 121°C for 10?min. Cytotoxicity of bacteriocin DU10 by methyl-thiazolyl-diphenyl-tetrazolium bromide assay showed that the viability of HT-29 and HeLa cells decreased 60?±?0.7% and 43?±?4.8%, respectively, in the presence of 3,200?AU/mL of bacteriocin. The strain withstood 0.3% w/v of bile oxgall and pH 2 affected the bacterial growth between 2 and 4?hr of incubation. Adhesion properties examined with HT-29 cell line showed 69.85% initial population of strain E. faecalis DU10, which was found to be strongly adhered to this cell line. These results conclude bacteriocin DU10 may be used as a potential biopreservative and E. faecalis DU10 may be used as a potential probiont to control Salmonella infections.     

Purification, amino acid sequence and characterization of Bacthuricin F4, a new bacteriocin produced by Bacillus thuringiensis

AIMS: Purification and characterization of a new bacteriocin, Bacthuricin F4 of Bacillus thuringiensis. METHODS AND RESULTS: A newly isolated B. thuringiensis subsp. kurstaki strain BUPM4, was shown to produce a novel bacteriocin named Bacthuricin F4. The highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. Bacthuricin F4 could be purified by a two-step procedure: ammonium sulphate precipitation of protein from culture supernatant followed by a reverse phase chromatography. Upon purification, the specific activity was increased 100-fold. This bacteriocin was heat-stable up to 70 degrees C and resisted up to pH 3.0. Bacthuricin F4 was sensitive to proteases demonstrating its proteinaceous nature. Its molecular mass, determined by mass spectrometry was 3160.05 Da. Direct N-terminal sequencing of Bacthuricin F4 revealed the following sequence: DWTXWSXL. The latter was unique in the databases. Bacthuricin F4 was active against Bacillus species while it had little or no effect on Gram-negative bacteria. CONCLUSIONS: A strain BUPM4 of B. thuringiensis subsp. kurstaki, was shown to produce a new bacteriocin named Bacthuricin F4 of both new molecular mass (3160.05 Da) and new amino acid terminal sequence. This is, to our knowledge, the first bacteriocin exhibiting such characteristics reported to be produced by B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The bacteriocin produced by the B. thuringiensis strain BUPM4 respond to both criteria of thermostability and stability to low pHs. Thus, it could be used for the control of the related species of Bacillus harmful for agricultural products.     

Identification, purification and characterization of laterosporulin, a novel bacteriocin produced by Brevibacillus sp. strain GI-9

Background

Bacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology.

Methodology/Findings

The strain GI-9 was identified as Brevibacillus sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database.

Conclusions

We have identified a bacteriocin producing strain GI-9, belonging to the genus Brevibacillus sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity.     

Conversion of squid pen by a novel strain Lactobacillus paracasei subsp. paracasei TKU010, and its application in antimicrobial and antioxidants activity

TKU010 was isolated from infant vomited milk and identified as Lactobacillus paracasei subsp. paracasei. TKU010 had desirable properties concerning its ability to withstand adverse conditions in the gastrointestinal tract. The hydrolysate of casein enhanced the growth of TKU010 most obviously (17.20-18.25 OD(660)), followed by the hydrolysate of SPP (16.00-15.06 OD(660)). Incubating with SPP, both the culture supernatant of TKU010 on the first day and the fourth day showed inhibitory activities on E. coli BCRC13086, F. oxysporum BCRC32121 and A. fumigatus BCRC30099. TKU010 culture supernatant (1% SPP) incubated for 3 days has high antioxidant activity; the DPPH scavenging ability was 75% per ml. Thus, TKU010 could be preferably used as a starter to produce fermented milk with possibly interesting organoleptic properties. Besides, we have shown that squid pen wastes can be utilized to generate a high value-added product, and have revealed its hidden potential in the production of biocontrol agents and functional foods.     

An inducible activator produced by a Serratia proteamaculans strain and its soybean growth-promoting activity under greenhouse conditions

Serratia proteamaculans 1-102 (1-102) promotes soybean-bradyrhizobia nodulation and growth, but the mechanism is unknown. After adding isoflavonoid inducers to 1-102 culture, an active peak with a retention time of about 105 min in the HPLC fractionation was isolated using a bioassay based on the stimulation of soybean seed germination. The plant growth-promoting activity of this material was compared with 1-102 culture (cells) and supernatant under greenhouse conditions. The activator was applied to roots in 83, 830 and 8300 HPLC microvolts (microV) per seedling when plants were inoculated with bradyrhizobia or sprayed onto the leaves in same concentrations at 20 d after inoculation. The root-applied activator, especially at 1 ml of 830 microV per seedling, enhanced soybean nodulation and growth at the same level as 1-102 culture under both optimal and sub-optimal root zone temperatures. Thus, this activator stimulating soybean seed germination is also responsible for the plant growth-promoting activity of 1-102 culture. However, when sprayed onto the leaves, the activator did not increase growth and in higher concentrations decreased average single leaf area. The results suggest that this inducible activator might be a lipo-chitooligosaccharide (LCO) analogue. LCOs act as rhizobia-to-legume signals stimulating root nodule formation. The activator could provide additional 'signal', increasing in the signal quality (the signal-to-noise ratio, SNR) of the plant-rhizobia signal exchange process.     

Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9

AIMS: To identify and characterize new bacteriocins from a collection of 41 strains belonging to 27 subspecies of Bacillus thuringiensis, and to evaluate the safety of the producers. METHODS AND RESULTS: Bacillus thuringiensis ssp. entomocidus HD9 produced in the culture supernatant an antimicrobial activity against Gram-positive bacteria including Listeria monocytogenes, one of four pathogenic Pseudomonas aeruginosa and several fungi. Production of the antibacterial activity, named entomocin 9, started during mid-logarithmic growth reaching its maximum at the early stationary phase. Entomocin 9 retained more than 72% of activity after incubation for 20 min at 121 degrees C. Activity was lost after proteinase K treatment, it was stable in a pH range between 3 and 9, and resistant to lyophilization. After partial purification with ammonium sulphate precipitation followed by gel-filtration and anion-exchange chromatography, an active protein of ca 12.4 kDa was isolated. The mode of action of entomocin 9 was bactericidal and caused cell lysis of growing cells. Despite the presence of a range of virulence related genes, including haemolysin BL, nonhaemolytic enterotoxin, cytotoxin K and several hydrolytic activities, B. thuringiensis HD9 was not toxic against Vero cells. CONCLUSIONS: Entomocin 9 is a novel heat-stable, bacteriocin produced by B. thuringiensis HD9. The absence of toxicity against Vero cells suggests the suitability of strain HD9 for a safe application in antimicrobial treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: New finding on entomocin 9 would make B. thuringiensis attractive in biotechnological applications as an antimicrobial agent in agriculture and food industry.     

Isolation and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi

Aims:  Screening and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi, a traditional Korean fermented vegetable.
Methods and Results:  A total of 1000 lactic acid bacteria were isolated from various Kimchi samples and screened for the production of bacteriocin. Pediocin K23-2, a bacteriocin produced by the Pediococcus pentosaceus K23-2 strain, showed strong inhibitory activity against Listeria monocytogenes . The bacteriocin activity remained unchanged after 15 min of heat treatment at 121°C or exposure to organic solvents; however, it diminished after treatment with proteolytic enzymes. The bacteriocin was maximally produced at 37°C, when the pH of the culture broth was maintained at 5·0 during the fermentation, although the optimum pH for growth was 7·0. The molecular weight of the bacteriocin was about 5 kDa according to a tricine SDS-PAGE analysis.
Conclusions:  Pediococcus pentosaceus K23-2 isolated from Kimchi produces a bacteriocin, which shares similar characteristics to the Class IIa bacteriocins. The bacteriocin is heat stable and shows wide antimicrobial activity against Gram-positive bacteria, especially L. monocytogenes .
Significance and Impact of the Study:  Pediocin K23-2 and pediocin K23-2-producing P. pentosaceus K23-2 could potentially be used in the food and feed industries as natural biopreservatives, and for probiotic application to humans or livestock.     

Detection and characterization of cerein 7, a new bacteriocin produced by Bacillus cereus with a broad spectrum of activity

A bacteriocin-producing strain of Bacillus cereus was identified and isolated from a soil sample. The bacteriocin could be purified by a two-step procedure: ammonium sulfate precipitation of culture supernatants followed by a butanol extraction step, the antibiotic was recovered from the organic phase. The peptidic nature of the bacteriocin was proven by its sensitivity to proteolytic enzymes; its molecular mass, determined by mass spectrometry, was 3940 Da; and its amino-terminal sequence (GWGDVL) is unique in the databases. The compound was active against most Gram-positive but not Gram-negative bacteria. This is to our knowledge the first bacteriocin with these characteristics reported to be produced by B. cereus.     

Influence of medium components and fermentation conditions on the production of bacteriocin(s) by Bacillus licheniformis AnBa9

Recently, antibacterial peptides are gaining more attention as an alternative therapeutics and food and other products from spoilage and deterioration. Antibacterial peptide producing strains were isolated from sediments of slaughterhouse sewage wastes. One among them, identified as Bacillus licheniformis inhibited the growth of several gram positive bacteria. Response surface methodology with central composite rotary design was used for optimization of fermentation medium and conditions for antibacterial peptide production. Lactose, NH(4)NO(3), yeast extract and NaCl and environmental factors such as pH, temperature and incubation period were selected as variables. Among ingredients, high concentration of yeast extract and NaCl had a positive effect on antibacterial peptide production and specific activity, respectively. Alkaline pH and high temperature favoured the production of antibacterial peptide by B. licheniformis AnBa9. Under optimized condition, B. licheniformis AnBa9 produced 25-fold higher production of antibacterial peptide than the un-optimized condition. Biochemical characteristics of the antibacterial peptides of B. licheniformis AnBa9 revealed that they are of bacteriocin type.     

Bactericidal activity of the membrane fraction isolated from phagocytes of mice and its stimulation by melittin

The membrane fraction prepared from mouse peritoneal exudate cells was incubated with mycobacteria, staphylococci, or E. coli in acetate buffer of pH 5.6 to follow the fate of viable bacilli. The membrane fraction exhibited bactericidal effect on mycobacteria and staphylococci, but not on E. coli. The activity to kill mycobacteria, as well as the endogenous phospholipase A2 activity, of the membrane fraction was markedly enhanced by melittin, a basic peptide from bee venom, and inhibited by indomethacin and EDTA. The role of the enzyme activity in the bactericidal activity was discussed.     

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

产紫青霉突变菌株Li-3-9的发酵条件优化及红色素安全性评价

本实验对前期研究得到的具有高产红色素能力的产紫青霉突变菌株Penicillium purpurogenum Li-3-9的发酵条件进行优化的同时作出了对红色素的安全性评价。在单因素试验的基础上,采用Box-Behnken试验设计优化了蔗糖浓度、酵母膏浓度和装液量对红色素色价的影响;并且研究了P. purpurogenum Li-3-9产生的红色素对斑马鱼胚胎的毒性效应。结果表明,最佳发酵条件为：蔗糖40g/L、酵母膏4g/L、装液量为45mL、接种量4%、培养温度32℃、初始pH值6.9、培养时间168h、摇床转速150r/min,红色素色价最高达到了11.4U/mL。通过计算斑马鱼胚胎致死率、心率、畸形率及孵化率可知,该色素无致死毒性,并且对斑马鱼胚胎发育具有一定的促进作用。     

Regulation of ADAMTS9 secretion and enzymatic activity by its propeptide

ADAMTS9 is a secreted, cell-surface-binding metalloprotease that cleaves the proteoglycans versican and aggrecan. Unlike most precursor proteins, the ADAMTS9 zymogen (pro-ADAMTS9) is resistant to intracellular processing. Instead, pro-ADAMTS9 is processed by furin at the cell surface. Here, we investigated the role of the ADAMTS9 propeptide in regulating its secretion and proteolytic activity. Removal of the propeptide abrogated secretion of the ADAMTS9 catalytic domain, and secretion was inefficiently restored by expression of the propeptide in trans. Substitution of Ala for Asn residues within each of three consensus N-linked glycosylation sites in the propeptide abrogated ADAMTS9 secretion. Thus, the propeptide is an intramolecular chaperone whose glycosylation is critical for secretion of the mature enzyme. In addition to two previously identified furin-processing sites (Arg74 downward arrow and Arg287 downward arrow) the ADAMTS9 propeptide was also furin-processed at Arg209. Substitution of Ala for Arg74, Arg209, and Arg287 resulted in secretion of an unprocessed zymogen. Unexpectedly, versican incubated with cells expressing this pro-ADAMTS9 was processed to a greater extent than when incubated with cells expressing wild-type, furin-processable ADAMTS9. Moreover, cells and medium treated with the proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone had greater versican-cleaving activity than untreated cells. Following furin processing of pro-ADAMTS9, propeptide fragments maintained a non-covalent association with the catalytic domain. Collectively, these observations suggest that, unlike other metalloproteases, furin processing of the ADAMTS9 propeptide reduces its catalytic activity. Thus, the propeptide is a key functional domain of ADAMTS9, mediating an unusual regulatory mechanism that may have evolved to ensure maximal activity of this protease at the cell surface.     

Detection and characterization of a bacteriocin, garviecin L1-5, produced by Lactococcus garvieae isolated from raw cow's milk

AIMS: The identification of a bacteriocin-producing lactococcal strain isolated from raw cow's milk is reported, along with production conditions, physical and chemical properties, and mode of action of the bacteriocin. METHODS and RESULTS: On the basis of resistance to clindamycin, species-specific PCR and amplification of the 16S-23S rDNA spacer region, the strain was identified as Lactococcus garvieae. Its bacteriocin, designated garviecin L1-5, was bactericidal against closely related species and strains of species from different genera, including Listeria monocytogenes and Clostridium spp. Garviecin L1-5 was shown to be proteinaceous by protease inactivation and was unaffected by heat treatments, also at low pH values. When amplifying known lactococcal bacteriocin genes using DNA from strain L1-5 as template, no amplification products were observed on the agarose gel. The molecular weight of garviecin L1-5 was about 2.5 kDa. As far as is known, no bacteriocins have been detected from Lactococcus garvieae. CONCLUSION: The general properties of garviecin L1-5 are characteristic of the low-molecular-weight bactericidal peptide group. SIGNIFICANCE AND IMPACT OF THE STUDY: The survey of micro-organisms for novel antimicrobial substances provided valuable information on their physiology, ecology and practical application.