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1.  Study of the Ecology of Fresh Sausages and Characterization of Populations of Lactic Acid Bacteria by Molecular Methods 
In this study, a polyphasic approach was used to study the ecology of fresh sausages and to characterize populations of lactic acid bacteria (LAB). The microbial profile of fresh sausages was monitored from the production day to the 10th day of storage at 4°C. Samples were collected on days 0, 3, 6, and 10, and culture-dependent and -independent methods of detection and identification were applied. Traditional plating and isolation of LAB strains, which were subsequently identified by molecular methods, and the application of PCR-denaturing gradient gel electrophoresis (DGGE) to DNA and RNA extracted directly from the fresh sausage samples allowed the study in detail of the changes in the bacterial and yeast populations during storage. Brochothrix thermosphacta and Lactobacillus sakei were the main populations present. In particular, B. thermosphacta was present throughout the process, as determined by both DNA and RNA analysis. Other bacterial species, mainly Staphylococcus xylosus, Leuconostoc mesenteroides, and L. curvatus, were detected by DGGE. Moreover, an uncultured bacterium and an uncultured Staphylococcus sp. were present, too. LAB strains isolated at day 0 were identified as Lactococcus lactis subsp. lactis, L. casei, and Enterococcus casseliflavus, and on day 3 a strain of Leuconostoc mesenteroides was identified. The remaining strains isolated belonged to L. sakei. Concerning the yeast ecology, only Debaryomyces hansenii was established in the fresh sausages. Capronia mansonii was initially present, but it was not detected after the first 3 days. At last, L. sakei isolates were characterized by randomly amplified polymorphic DNA PCR and repetitive DNA element PCR. The results obtained underlined how different populations took over at different steps of the process. This is believed to be the result of the selection of the particular population, possibly due to the low storage temperature employed.
PMCID: PMC383013  PMID: 15066777
2.  Antagonistic lactic acid bacteria isolated from goat milk and identification of a novel nisin variant Lactococcus lactis 
BMC Microbiology  2014;14:36.
The raw goat milk microbiota is considered a good source of novel bacteriocinogenic lactic acid bacteria (LAB) strains that can be exploited as an alternative for use as biopreservatives in foods. The constant demand for such alternative tools justifies studies that investigate the antimicrobial potential of such strains.
The obtained data identified a predominance of Lactococcus and Enterococcus strains in raw goat milk microbiota with antimicrobial activity against Listeria monocytogenes ATCC 7644. Enzymatic assays confirmed the bacteriocinogenic nature of the antimicrobial substances produced by the isolated strains, and PCR reactions detected a variety of bacteriocin-related genes in their genomes. Rep-PCR identified broad genetic variability among the Enterococcus isolates, and close relations between the Lactococcus strains. The sequencing of PCR products from nis-positive Lactococcus allowed the identification of a predicted nisin variant not previously described and possessing a wide inhibitory spectrum.
Raw goat milk was confirmed as a good source of novel bacteriocinogenic LAB strains, having identified Lactococcus isolates possessing variations in their genomes that suggest the production of a nisin variant not yet described and with potential for use as biopreservatives in food due to its broad spectrum of action.
PMCID: PMC3930553  PMID: 24521354
3.  Cloning and Heterologous Production of Hiracin JM79, a Sec-Dependent Bacteriocin Produced by Enterococcus hirae DCH5, in Lactic Acid Bacteria and Pichia pastoris▿  
Hiracin JM79 (HirJM79), a Sec-dependent bacteriocin produced by Enterococcus hirae DCH5, was cloned and produced in Lactococcus lactis, Lactobacillus sakei, Enterococcus faecium, Enterococcus faecalis, and Pichia pastoris. For heterologous production of HirJM79 in lactic acid bacteria (LAB), the HirJM79 structural gene (hirJM79), with or without the HirJM79 immunity gene (hiriJM79), was cloned into the plasmid pMG36c under the control of the constitutive promoter P32 and into the plasmid pNZ8048 under the control of the inducible PNisA promoter. For the production of HirJM79 in P. pastoris, the gene encoding the mature HirJM79 protein was cloned into the pPICZαA expression vector. The recombinant plasmids permitted the production of biologically active HirJM79 in the supernatants of L. lactis IL1403, L. lactis NZ9000, L. sakei Lb790, E. faecalis JH2-2, and P. pastoris X-33, the coproduction of HirJM79 and nisin A in L. lactis DPC5598, and the coproduction of HirJM79 and enterocin P in E. faecium L50/14-2. All recombinant LAB produced larger quantities of HirJM79 than E. hirae DCH5, although the antimicrobial activities of most transformants were lower than that predicted from their production of HirJM79. The synthesis, processing, and secretion of HirJM79 proceed efficiently in recombinant LAB strains and P. pastoris.
PMCID: PMC2293139  PMID: 18310424
4.  Simultaneous Production of Biosurfactants and Bacteriocins by Probiotic Lactobacillus casei MRTL3 
Lactic acid bacteria (LAB) are ubiquitous and well-known commensal bacteria in the human and animal microflora. LAB are extensively studied and used in a variety of industrial and food fermentations. They are widely used for humans and animals as adjuvants, probiotic formulation, and dietary supplements and in other food fermentation applications. In the present investigation, LAB were isolated from raw milk samples collected from local dairy farms of Haryana, India. Further, the isolates were screened for simultaneous production of biosurfactants and bacteriocins. Biosurfactant produced was found to be a mixture of lipid and sugar similar to glycolipids. The bacteriocin obtained was found to be heat stable (5 min at 100°C). Further, DNA of the strain was extracted and amplified by the 16S rRNA sequencing using universal primers. The isolate Lactobacillus casei MRTL3 was found to be a potent biosurfactant and bacteriocin producer. It seems to have huge potential for food industry as a biopreservative and/or food ingredient.
PMCID: PMC3941777  PMID: 24669225
5.  Isolation and characteristics of lactic acid bacteria isolated from ripe mulberries in Taiwan 
Brazilian Journal of Microbiology  2010;41(4):916-921.
The objective of this study was to isolate, characterize, and identify lactic acid bacteria (LAB) from ripe mulberries collected in Taiwan. Ripe mulberry samples were collected at five mulberry farms, located in different counties of Taiwan. Eighty-eight acid-producing cultures were isolated from these samples, and isolates were divided into classes first by phenotype, then into groups by restriction fragment length polymorphism (RFLP) analysis and sequencing of 16S ribosomal DNA (rDNA). Phenotypic and biochemical characteristics led to identification of four bacterial groups (A to D). Weissella cibaria was the most abundant type of LAB distributed in four mulberry farms, and Lactobacillus plantarum was the most abundant LAB found in the remaining farm. Ten W. cibaria and one Lactococcus lactis subsp. lactis isolate produced bacteriocins against the indicator strain Lactobacillus sakei JCM 1157T. These results suggest that various LAB are distributed in ripe mulberries and W. cibaria was the most abundant LAB found in this study.
PMCID: PMC3769767  PMID: 24031571
lactic acid bacteria; mulberry; Weissella cibaria; bacteriocin; Taiwan
6.  Incidence of Bacteriocins Produced by Food-Related Lactic Acid Bacteria Active towards Oral Pathogens 
In the present study we investigated the incidence of bacteriocins produced by 236 lactic acid bacteria (LAB) food isolates against pathogenic or opportunistic pathogenic oral bacteria. This set of LAB contained several strains (≥17%) producing bacteriocins active against food-related bacteria. Interestingly only Streptococcus macedonicus ACA-DC 198 was able to inhibit the growth of Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii, while Lactobacillus fermentum ACA-DC 179 and Lactobacillus plantarun ACA-DC 269 produced bacteriocins solely against Streptococcus oralis. Thus, the percentage of strains that were found to produce bacteriocins against oral bacteria was ~1.3%. The rarity of bacteriocins active against oral LAB pathogens produced by food-related LAB was unexpected given their close phylogenetic relationship. Nevertheless, when tested in inhibition assays, the potency of the bacteriocin(s) of S. macedonicus ACA-DC 198 against the three oral streptococci was high. Fourier-transform infrared spectroscopy combined with principal component analysis revealed that exposure of the target cells to the antimicrobial compounds caused major alterations of key cellular constituents. Our findings indicate that bacteriocins produced by food-related LAB against oral LAB may be rare, but deserve further investigation since, when discovered, they can be effective antimicrobials.
PMCID: PMC3634443  PMID: 23443163
lactic acid bacteria; food; oral; Streptococcus; bacteriocin; FT-IR
7.  Macedocin, a Food-Grade Lantibiotic Produced by Streptococcus macedonicus ACA-DC 198 
Applied and Environmental Microbiology  2002;68(12):5891-5903.
Streptococcus macedonicus ACA-DC 198, a strain isolated from Greek Kasseri cheese, produces a food-grade lantibiotic named macedocin. Macedocin has a molecular mass of 2,794.76 ± 0.42 Da, as determined by electrospray mass spectrometry. Partial N-terminal sequence analysis revealed 22 amino acid residues that correspond with the amino acid sequence of the lantibiotics SA-FF22 and SA-M49, both of which were isolated from the pathogen Streptococcus pyogenes. Macedocin inhibits a broad spectrum of lactic acid bacteria, as well as several food spoilage and pathogenic bacteria, including Clostridium tyrobutyricum. It displays a bactericidal effect towards the most sensitive indicator strain, Lactobacillus sakei subsp. sakei LMG 13558T, while the producer strain itself displays autoinhibition when it is grown under conditions that do not favor bacteriocin production. Macedocin is active at pHs between 4.0 and 9.0, and it retains activity even after incubation for 20 min at 121°C with 1 atm of overpressure. Inhibition of macedocin by proteolytic enzymes is variable.
PMCID: PMC134371  PMID: 12450808
8.  Isolation and Identification of the Microbiota of Danish Farmhouse and Industrially Produced Surface-Ripened Cheeses 
Microbial Ecology  2012;65(3):602-615.
For studying the microbiota of four Danish surface-ripened cheeses produced at three farmhouses and one industrial dairy, both a culture-dependent and culture-independent approach were used. After dereplication of the initial set of 433 isolates by (GTG)5-PCR fingerprinting, 217 bacterial and 25 yeast isolates were identified by sequencing of the 16S rRNA gene or the D1/D2 domain of the 26S rRNA gene, respectively. At the end of ripening, the cheese core microbiota of the farmhouse cheeses consisted of the mesophilic lactic acid bacteria (LAB) starter cultures Lactococcus lactis subsp. lactis and Leuconostoc mesenteorides as well as non-starter LAB including different Lactobacillus spp. The cheese from the industrial dairy was almost exclusively dominated by Lb. paracasei. The surface bacterial microbiota of all four cheeses were dominated by Corynebacterium spp. and/or Brachybacterium spp. Brevibacterium spp. was found to be subdominant compared to other bacteria on the farmhouse cheeses, and no Brevibacterium spp. was found on the cheese from the industrial dairy, even though B. linens was used as surface-ripening culture. Moreover, Gram-negative bacteria identified as Alcalignes faecalis and Proteus vulgaris were found on one of the farmhouse cheeses. The surface yeast microbiota consisted primarily of one dominating species for each cheese. For the farmhouse cheeses, the dominant yeast species were Yarrowia lipolytica, Geotrichum spp. and Debaryomyces hansenii, respectively, and for the cheese from the industrial dairy, D. hansenii was the dominant yeast species. Additionally, denaturing gradient gel electrophoresis (DGGE) analysis revealed that Streptococcus thermophilus was present in the farmhouse raw milk cheese analysed in this study. Furthermore, DGGE bands corresponding to Vagococcus carniphilus, Psychrobacter spp. and Lb. curvatus on the cheese surfaces indicated that these bacterial species may play a role in cheese ripening.
PMCID: PMC3621994  PMID: 23224222
9.  Antibacterial Activities of Nisin Z Encapsulated in Liposomes or Produced In Situ by Mixed Culture during Cheddar Cheese Ripening 
Applied and Environmental Microbiology  2002;68(11):5607-5619.
This study investigated both the activity of nisin Z, either encapsulated in liposomes or produced in situ by a mixed starter, against Listeria innocua, Lactococcus spp., and Lactobacillus casei subsp. casei and the distribution of nisin Z in a Cheddar cheese matrix. Nisin Z molecules were visualized using gold-labeled anti-nisin Z monoclonal antibodies and transmission electron microscopy (immune-TEM). Experimental Cheddar cheeses were made using a nisinogenic mixed starter culture, containing Lactococcus lactis subsp. lactis biovar diacetylactis UL 719 as the nisin producer and two nisin-tolerant lactococcal strains and L. casei subsp. casei as secondary flora, and ripened at 7°C for 6 months. In some trials, L. innocua was added to cheese milk at 105 to 106 CFU/ml. In 6-month-old cheeses, 90% of the initial activity of encapsulated nisin (280 ± 14 IU/g) was recovered, in contrast to only 12% for initial nisin activity produced in situ by the nisinogenic starter (300 ± 15 IU/g). During ripening, immune-TEM observations showed that encapsulated nisin was located mainly at the fat/casein interface and/or embedded in whey pockets while nisin produced by biovar diacetylactis UL 719 was uniformly distributed in the fresh cheese matrix but concentrated in the fat area as the cheeses aged. Cell membrane in lactococci appeared to be the main nisin target, while in L. casei subsp. casei and L. innocua, nisin was more commonly observed in the cytoplasm. Cell wall disruption and digestion and lysis vesicle formation were common observations among strains exposed to nisin. Immune-TEM observations suggest several modes of action for nisin Z, which may be genus and/or species specific and may include intracellular target-specific activity. It was concluded that nisin-containing liposomes can provide a powerful tool to improve nisin stability and availability in the cheese matrix.
PMCID: PMC129882  PMID: 12406756
10.  Isolation of Pediococcus acidilactici Kp10 with ability to secrete bacteriocin-like inhibitory substance from milk products for applications in food industry 
BMC Microbiology  2012;12:260.
Lactic acid bacteria (LAB) can be isolated from traditional milk products. LAB that secrete substances that inhibit pathogenic bacteria and are resistant to acid, bile, and pepsin but not vancomycin may have potential in food applications.
LAB isolated from a range of traditional fermented products were screened for the production of bacteriocin-like inhibitory substances. A total of 222 LAB strains were isolated from fermented milk products in the form of fresh curds, dried curds, and ghara (a traditional flavor enhancer prepared from whey), and fermented cocoa bean. Eleven LAB isolates that produced antimicrobial substances were identified as Lactococcus lactis, Lactobacillus plantarum, and Pediococcus acidilactici strains by biochemical methods and 16S rDNA gene sequencing. Of these, the cell-free supernatant of Kp10 (P. acidilactici) most strongly inhibited Listeria monocytogenes. Further analysis identified the antimicrobial substance produced by Kp10 as proteinaceous in nature and active over a wide pH range. Kp10 (P. acidilactici) was found to be catalase-negative, able to produce β-galactosidase, resistant to bile salts (0.3%) and acidic conditions (pH 3), and susceptible to most antibiotics.
Traditionally prepared fermented milk products are good sources of LAB with characteristics suitable for industrial applications. The isolate Kp10 (P. acidilactici) shows potential for the production of probiotic and functional foods.
PMCID: PMC3571982  PMID: 23153191
Lactic acid bacteria; Pediococcus acidilactici; Bacteriocin-like inhibitory substance; Listeria monocytogenes; Fermentation; Identification
11.  Diversity, Dynamics, and Activity of Bacterial Communities during Production of an Artisanal Sicilian Cheese as Evaluated by 16S rRNA Analysis†  
The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.
PMCID: PMC123848  PMID: 11916708
12.  Prevalence of antibiotic resistance in lactic acid bacteria isolated from the faeces of broiler chicken in Malaysia 
Gut Pathogens  2014;6:1.
Probiotics are commonly used as feed additive to substitute antibiotic as growth promoter in animal farming. Probiotic consists of lactic acid bacteria (LAB), which enhance the growth and health of the animal. Probiotic also have higher possibility to become pathogenic bacteria that may carry antibiotic resistant gene that can be transmitted to other LAB species. The aim of this study was to identify the LAB species in the faeces of broiler chicken and to determine the prevalence of antibiotic resistant in LAB of broiler chicken.
Sixty faeces samples were collected from wet markets located in Klang Valley of Malaysia for the isolation of LAB using de-Mann Rogosa Sharpe medium. Thirteen species of LAB were obtained in this study and the identification of LAB was performed by using API test kit on the basis of carbohydrate fermentation profile. Antibiotic susceptibility assay was then carried out to determine the prevalence of LAB antibiotic resistance.
Lactococcus lactis subsp lactis was found in nine out of sixty faecal samples. Lactobacillus paracasei was the second common LAB species isolated from chicken faecal. No significant difference (P > 0.05) was found between the occurrence of Lactobacillus brevis, Lactobacillus curvatus, Lactobacillus plantarum, Leuconostoc lactis mesenteroides subsp mesenteroides/dectranium and Pediococcus pentosaceus isolated from 5 different locations. Most of the isolated LAB was resistant to antibiotic and high variability of the antibiotic resistance was observed among the LAB against 15 types of antibiotics. Penicillin, amoxicillin, chloramphenicol, and ampicillin had significant higher (P< 0.05) inhibitory zone than nalidixic acid, gentamycin, sulphamethoxazole, kanamycin, and streptomycin.
Many species of LAB were isolated from the faecal samples of broiler chicken that resistance to the common antibiotics used in the farm. The development of resistant against antibiotics in LAB can be attributed to the long term exposure of antibiotic as growth promoter and therapeutic agents. Thus, it is essential to advise farmer the safety measure of antibiotic application in animal farming. Additionally, the supplementation of probiotic in animal feeding also needs more attention and close monitoring.
PMCID: PMC3902413  PMID: 24447766
Lactic acid bacteria; Antibiotic resistance; Broiler chicken
13.  Phylogenomic reconstruction of lactic acid bacteria: an update 
Lactic acid bacteria (LAB) are important in the food industry for the production of fermented food products and in human health as commensals in the gut. However, the phylogenetic relationships among LAB species remain under intensive debate owing to disagreements among different data sets.
We performed a phylogenetic analysis of LAB species based on 232 genes from 28 LAB genome sequences. Regardless of the tree-building methods used, combined analyses yielded an identical, well-resolved tree topology with strong supports for all nodes. The LAB species examined were divided into two groups. Group 1 included families Enterococcaceae and Streptococcaceae. Group 2 included families Lactobacillaceae and Leuconostocaceae. Within Group 2, the LAB species were divided into two clades. One clade comprised of the acidophilus complex of genus Lactobacillus and two other species, Lb. sakei and Lb. casei. In the acidophilus complex, Lb. delbrueckii separated first, while Lb. acidophilus/Lb. helveticus and Lb. gasseri/Lb. johnsonii were clustered into a sister group. The other clade within Group 2 consisted of the salivarius subgroup, including five species, Lb. salivarius, Lb. plantarum, Lb. brevis, Lb. reuteri, Lb. fermentum, and the genera Pediococcus, Oenococcus, and Leuconostoc. In this clade, Lb. salivarius was positioned most basally, followed by two clusters, one corresponding to Lb. plantarum/Lb. brevis pair and Pediococcus, and the other including Oenococcus/Leuconostoc pair and Lb. reuteri/Lb. fermentum pair. In addition, phylogenetic utility of the 232 genes was analyzed to identify those that may be more useful than others. The genes identified as useful were related to translation and ribosomal structure and biogenesis (TRSB), and a three-gene set comprising genes encoding ultra-violet resistance protein B (uvrB), DNA polymerase III (polC) and penicillin binding protein 2B (pbpB).
Our phylogenomic analyses provide important insights into the evolution and diversification of LAB species, and also revealed the phylogenetic utility of several genes. We infer that the occurrence of multiple, independent adaptation events in LAB species, have resulted in their occupation of various habitats. Further analyses of more genes from additional, representative LAB species are needed to reveal the molecular mechanisms underlying adaptation of LAB species to various environmental niches.
PMCID: PMC3024227  PMID: 21194491
14.  Antimicrobial and Safety Properties of Lactobacilli Isolated from two Cameroonian Traditional Fermented Foods 
Scientia Pharmaceutica  2011;80(1):189-203.
Twenty-one Lactobacillus isolates from “Sha’a” (a maize – based fermented beverage) and “Kossam” (traditionally fermented cow milk) were selected in accordance with their antagonistic activities and tested for their bacteriocinogenic potential as well as safety properties. These isolates were preliminarily identified as Lactobacillus plantarum (62%), Lactobacillus rhamnosus (24%), Lactobacillus fermentum (10%) and Lactobacillus coprophilus (4%) based on phenotypic characteristics and rep-PCR genomic fingerprinting. Twelve (57.1%) out of the 21 strains tested were found to be bacteriocin producers, as revealed by the sensitivity of their antimicrobial substances to proteolytic enzymes (Trypsin, Proteinase K) and inhibition of other Lactobacillus spp. These bacteriocinogenic strains showed no positive haemolytic and gelatinase activities and proved to be sensitive to penicillin G, ampicillin, tetracycline, erythromycin, amoxicillin, chloramphenicol, co-trimoxazole and doxycyclin, but resistant to ciprofloxacin and gentamicin. The bacteriocins showed a broad inhibitory activity against Gram-positive and Gram-negative pathogenic bacteria, several of which are classified as especially dangerous by the World Health Organization, as well as Multidrug-resistant strains. These include Staphylococcus aureus, Salmonella enterica subsp. enterica serovare Typhi, Bacillus cereus, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Shigella flexneri. These Lactobacillus strains are promising candidates for use as protective cultures in food fermentation.
PMCID: PMC3293358  PMID: 22396914
Sha’a; Kossam; Lactobacillus spp.; Rep-PCR; Safety properties; Antimicrobial activity
15.  Inhibitory activity of Lactobacillus plantarum LMG P-26358 against Listeria innocua when used as an adjunct starter in the manufacture of cheese 
Microbial Cell Factories  2011;10(Suppl 1):S7.
Lactobacillus plantarum LMG P-26358 isolated from a soft French artisanal cheese produces a potent class IIa bacteriocin with 100% homology to plantaricin 423 and bacteriocidal activity against Listeria innocua and Listeria monocytogenes. The bacteriocin was found to be highly stable at temperatures as high as 100°C and pH ranges from 1-10. While this relatively narrow spectrum bacteriocin also exhibited antimicrobial activity against species of enterococci, it did not inhibit dairy starters including lactococci and lactobacilli when tested by well diffusion assay (WDA). In order to test the suitability of Lb. plantarum LMG P-26358 as an anti-listerial adjunct with nisin-producing lactococci, laboratory-scale cheeses were manufactured. Results indicated that combining Lb. plantarum LMG P-26358 (at 108 colony forming units (cfu)/ml) with a nisin producer is an effective strategy to eliminate the biological indicator strain, L. innocua. Moreover, industrial-scale cheeses also demonstrated that Lb. plantarum LMG P-26358 was much more effective than the nisin producer alone for protection against the indicator. MALDI-TOF mass spectrometry confirmed the presence of plantaricin 423 and nisin in the appropriate cheeses over an 18 week ripening period. A spray-dried fermentate of Lb. plantarum LMG P-26358 also demonstrated potent anti-listerial activity in vitro using L. innocua. Overall, the results suggest that Lb. plantarum LMG P-26358 is a suitable adjunct for use with nisin-producing cultures to improve the safety and quality of dairy products.
PMCID: PMC3231933  PMID: 21995443
16.  Complete Sequencing and Pan-Genomic Analysis of Lactobacillus delbrueckii subsp. bulgaricus Reveal Its Genetic Basis for Industrial Yogurt Production 
PLoS ONE  2011;6(1):e15964.
Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus) is an important species of Lactic Acid Bacteria (LAB) used for cheese and yogurt fermentation. The genome of Lb. bulgaricus 2038, an industrial strain mainly used for yogurt production, was completely sequenced and compared against the other two ATCC collection strains of the same subspecies. Specific physiological properties of strain 2038, such as lysine biosynthesis, formate production, aspartate-related carbon-skeleton intermediate metabolism, unique EPS synthesis and efficient DNA restriction/modification systems, are all different from those of the collection strains that might benefit the industrial production of yogurt. Other common features shared by Lb. bulgaricus strains, such as efficient protocooperation with Streptococcus thermophilus and lactate production as well as well-equipped stress tolerance mechanisms may account for it being selected originally for yogurt fermentation industry. Multiple lines of evidence suggested that Lb. bulgaricus 2038 was genetically closer to the common ancestor of the subspecies than the other two sequenced collection strains, probably due to a strict industrial maintenance process for strain 2038 that might have halted its genome decay and sustained a gene network suitable for large scale yogurt production.
PMCID: PMC3022021  PMID: 21264216
17.  Proteins of novel lactic acid bacteria from Apis mellifera mellifera: an insight into the production of known extra-cellular proteins during microbial stress 
BMC Microbiology  2013;13:235.
Lactic acid bacteria (LAB) has been considered a beneficial bacterial group, found as part of the microbiota of diverse hosts, including humans and various animals. However, the mechanisms of how hosts and LAB interact are still poorly understood. Previous work demonstrates that 13 species of Lactobacillus and Bifidobacterium from the honey crop in bees function symbiotically with the honeybee. They protect each other, their hosts, and the surrounding environment against severe bee pathogens, bacteria, and yeasts. Therefore, we hypothesized that these LAB under stress, i.e. in their natural niche in the honey crop, are likely to produce bioactive substances with antimicrobial activity.
The genomic analysis of the LAB demonstrated varying genome sizes ranging from 1.5 to 2.2 mega-base pairs (Mbps) which points out a clear difference within the protein gene content, as well as specialized functions in the honeybee microbiota and their adaptation to their host. We demonstrate a clear variation between the secreted proteins of the symbiotic LAB when subjected to microbial stressors. We have identified that 10 of the 13 LAB produced extra-cellular proteins of known or unknown function in which some are arranged in interesting putative operons that may be involved in antimicrobial action, host interaction, or biofilm formation. The most common known extra-cellular proteins secreted were enzymes, DNA chaperones, S-layer proteins, bacteriocins, and lysozymes. A new bacteriocin may have been identified in one of the LAB symbionts while many proteins with unknown functions were produced which must be investigated further.
The 13 LAB symbionts likely play different roles in their natural environment defending their niche and their host and participating in the honeybee’s food production. These roles are partly played through producing extracellular proteins on exposure to microbial stressors widely found in natural occurring flowers. Many of these secreted proteins may have a putative antimicrobial function. In the future, understanding these processes in this complicated environment may lead to novel applications of honey crop LAB proteins.
PMCID: PMC4015849  PMID: 24148670
Lactic acid bacteria; Symbionts; Microbial stress; Proteomics; Honeybee
18.  Identification of Lactic Acid Bacteria from Chili Bo, a Malaysian Food Ingredient 
Ninety-two strains of lactic acid bacteria (LAB) were isolated from a Malaysian food ingredient, chili bo, stored for up to 25 days at 28°C with no benzoic acid (product A) or with 7,000 mg of benzoic acid kg−1 (product B). The strains were divided into eight groups by traditional phenotypic tests. A total of 43 strains were selected for comparison of their sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) whole-cell protein patterns with a SDS-PAGE database of LAB. Isolates from product A were identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus farciminis, Pediococcus acidilactici, Enterococcus faecalis, and Weissella confusa. Five strains belonging to clusters which could not be allocated to existing species by SDS-PAGE were further identified by 16S rRNA sequence comparison. One strain was distantly related to the Lactobacillus casei/Pediococcus group. Two strains were related to Weissella at the genus or species level. Two other strains did not belong to any previously described 16S rRNA group of LAB and occupied an intermediate position between the L. casei/Pediococcus group and the Weissella group and species of Carnobacterium. The latter two strains belong to the cluster of LAB that predominated in product B. The incidence of new species and subspecies of LAB in chili bo indicate the high probability of isolation of new LAB from certain Southeast Asian foods. None of the isolates exhibited bacteriocin activity against L. plantarum ATCC 14917 and LMG 17682.
PMCID: PMC91067  PMID: 9925588
19.  Antimicrobial activity, antibiotic susceptibility and virulence factors of Lactic Acid Bacteria of aquatic origin intended for use as probiotics in aquaculture 
BMC Microbiology  2013;13:15.
The microorganisms intended for use as probiotics in aquaculture should exert antimicrobial activity and be regarded as safe not only for the aquatic hosts but also for their surrounding environments and humans. The objective of this work was to investigate the antimicrobial/bacteriocin activity against fish pathogens, the antibiotic susceptibility, and the prevalence of virulence factors and detrimental enzymatic activities in 99 Lactic Acid Bacteria (LAB) (59 enterococci and 40 non-enterococci) isolated from aquatic animals regarded as human food.
These LAB displayed a broad antimicrobial/bacteriocin activity against the main Gram-positive and Gram-negative fish pathogens. However, particular safety concerns based on antibiotic resistance and virulence factors were identified in the genus Enterococcus (86%) (Enterococcus faecalis, 100%; E. faecium, 79%). Antibiotic resistance was also found in the genera Weissella (60%), Pediococcus (44%), Lactobacillus (33%), but not in leuconostocs and lactococci. Antibiotic resistance genes were found in 7.5% of the non-enterococci, including the genera Pediococcus (12.5%) and Weissella (6.7%). One strain of both Pediococcus pentosaceus and Weissella cibaria carried the erythromycin resistance gene mef(A/E), and another two P. pentosaceus strains harboured lnu(A) conferring resistance to lincosamides. Gelatinase activity was found in E. faecalis and E. faecium (71 and 11%, respectively), while a low number of E. faecalis (5%) and none E. faecium exerted hemolytic activity. None enterococci and non-enterococci showed bile deconjugation and mucin degradation abilities, or other detrimental enzymatic activities.
To our knowledge, this is the first description of mef(A/E) in the genera Pediococcus and Weissella, and lnu(A) in the genus Pediococcus. The in vitro subtractive screening presented in this work constitutes a valuable strategy for the large-scale preliminary selection of putatively safe LAB intended for use as probiotics in aquaculture.
PMCID: PMC3574848  PMID: 23347637
Lactic Acid Bacteria; Aquatic animals; Aquaculture probiotics; Anti-fish pathogens activity; Antibiotic resistance and virulence factors; Qualified Presumption of Safety
20.  Combined Effect of High-Pressure Treatments and Bacteriocin-Producing Lactic Acid Bacteria on Inactivation of Escherichia coli O157:H7 in Raw-Milk Cheese 
The effect of high-pressure (HP) treatments combined with bacteriocins of lactic acid bacteria (LAB) produced in situ on the survival of Escherichia coli O157:H7 in cheese was investigated. Cheeses were manufactured from raw milk inoculated with E. coli O157:H7 at approximately 105 CFU/ml. Seven different bacteriocin-producing LAB were added at approximately 106 CFU/ml as adjuncts to the starter. Cheeses were pressurized on day 2 or 50 at 300 MPa for 10 min or 500 MPa for 5 min, at 10°C in both cases. After 60 days, E. coli O157:H7 counts in cheeses manufactured without bacteriocin-producing LAB and not pressurized were 5.1 log CFU/g. A higher inactivation of E. coli O157:H7 was achieved in cheeses without bacteriocin-producing LAB when 300 MPa was applied on day 50 (3.8-log-unit reduction) than if applied on day 2 (1.3-log-unit reduction). Application of 500 MPa eliminated E. coli O157:H7 in 60-day-old cheeses. Cheeses made with bacteriocin-producing LAB and not pressurized showed a slight reduction of the pathogen. Pressurization at 300 MPa on day 2 and addition of lacticin 481-, nisin A-, bacteriocin TAB 57-, or enterocin AS-48-producing LAB were synergistic and reduced E. coli O157:H7 counts to levels below 2 log units in 60-day-old cheeses. Pressurization at 300 MPa on day 50 and addition of nisin A-, bacteriocin TAB 57-, enterocin I-, or enterocin AS-48-producing LAB completely inactivated E. coli O157:H7 in 60-day-old cheeses. The application of reduced pressures combined with bacteriocin-producing LAB is a feasible procedure to improve cheese safety.
PMCID: PMC1168985  PMID: 16000741
21.  Functional Screening of Antibiotic Resistance Genes from a Representative Metagenomic Library of Food Fermenting Microbiota 
BioMed Research International  2014;2014:290967.
Lactic acid bacteria (LAB) represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR) determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest.
PMCID: PMC4163480  PMID: 25243126
22.  Lactobacillus plantarum gene clusters encoding putative cell-surface protein complexes for carbohydrate utilization are conserved in specific gram-positive bacteria 
BMC Genomics  2006;7:126.
Genomes of gram-positive bacteria encode many putative cell-surface proteins, of which the majority has no known function. From the rapidly increasing number of available genome sequences it has become apparent that many cell-surface proteins are conserved, and frequently encoded in gene clusters or operons, suggesting common functions, and interactions of multiple components.
A novel gene cluster encoding exclusively cell-surface proteins was identified, which is conserved in a subgroup of gram-positive bacteria. Each gene cluster generally has one copy of four new gene families called cscA, cscB, cscC and cscD. Clusters encoding these cell-surface proteins were found only in complete genomes of Lactobacillus plantarum, Lactobacillus sakei, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Lactococcus lactis ssp lactis and Bacillus cereus and in incomplete genomes of L. lactis ssp cremoris, Lactobacillus casei, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillius brevis, Oenococcus oeni, Leuconostoc mesenteroides, and Bacillus thuringiensis. These genes are neither present in the genomes of streptococci, staphylococci and clostridia, nor in the Lactobacillus acidophilus group, suggesting a niche-specific distribution, possibly relating to association with plants. All encoded proteins have a signal peptide for secretion by the Sec-dependent pathway, while some have cell-surface anchors, novel WxL domains, and putative domains for sugar binding and degradation. Transcriptome analysis in L. plantarum shows that the cscA-D genes are co-expressed, supporting their operon organization. Many gene clusters are significantly up-regulated in a glucose-grown, ccpA-mutant derivative of L. plantarum, suggesting catabolite control. This is supported by the presence of predicted CRE-sites upstream or inside the up-regulated cscA-D gene clusters.
We propose that the CscA, CscB, CscC and CscD proteins form cell-surface protein complexes and play a role in carbon source acquisition. Primary occurrence in plant-associated gram-positive bacteria suggests a possible role in degradation and utilization of plant oligo- or poly-saccharides.
PMCID: PMC1534035  PMID: 16723015
23.  Antibacterial activity of Enterococcus faecium derived from Koopeh cheese against Listeria monocytogenes in probiotic ultra-filtrated cheese 
Viability of probiotic bacteria in food during maintenance and time of consuming in food has become a challenge in food hygiene and technology and is important for representing their beneficial health effects. The aim of this study was to determine the survival of probiotic Enterococcus faecium derived from Koopeh cheese added to industrial Iranian ultra-filtrated (UF) cheese and screening for antimicrobial activity of Enterococcus faecium against Listeria monocytogenes during two months of cheese ripening. Physiochemical and standard microbial methods were used for isolation of Enterococcus strains in cheese samples. The initial number of lactic acid bacteria (LAB) as starter culture was 6 Log g-1 in control samples. The counts started to decrease slightly after day seven (p < 0.05) and dropped to 5 Log g-1 at the end of 60 days. The count of LAB in the test groups decreased to 11 Log g-1 on the day 60 of ripening. The number of Enterococcus faecium was 6 Log g-1 on the day 60. The count of Listeria monocytogenes after 60 days of ripening in blank sample decreased 1 Log but in test samples with protective strain decreased 3 Log in 30 days and reached to zero at 45 days. There were not significant (p < 0.05) changes in chemical parameters such as fat, protein and total solid of UF cheese treatment groups. The results showed that Enterococcus faecium of Koopeh cheese was suitable for development of an acceptable probiotic UF cheese and could be adapted to industrial production of UF cheese.
PMCID: PMC4279643  PMID: 25568714
Enterococcus faecium; Listeria monocytogenes; Probiotic; Ultra-filtrated cheese
24.  Production of bacteriocin-like substances by lactic acid bacteria isolated from regional ovine cheese 
Brazilian Journal of Microbiology  2010;41(4):1009-1018.
Lactic acid bacteria (LAB) were isolated from ovine milk and cheeses manufactured in the South Region of Brazil. Among 112 bacterial isolates investigated, 59 were chosen through a screening for LAB. Among these 59 strains of LAB, 21% showed antimicrobial, proteolytic and lipolytic activities. Based on this screening, Lactobacillus plantarum LCN 17 and Lactobacillus rhamnosus LCN 43 were selected and tested for the production of bacteriocin-like substances (BLS). The BLS produced by both isolates showed antimicrobial activity against Listeria monocytogenes, whereas that produced by L. plantarum LCN 17 presented higher stability to different temperature, pH and enzyme treatments. These strains present potential for production of BLS, and for use as starter cultures.
PMCID: PMC3769745  PMID: 24031581
lactic acid bacteria; bacteriocin-like substances; ewe’s raw milk; Brazilian ovine cheese
25.  The Cyclic Antibacterial Peptide Enterocin AS-48: Isolation, Mode of Action, and Possible Food Applications 
Enterocin AS-48 is a circular bacteriocin produced by Enterococcus. It contains a 70 amino acid-residue chain circularized by a head-to-tail peptide bond. The conformation of enterocin AS-48 is arranged into five alpha-helices with a compact globular structure. Enterocin AS-48 has a wide inhibitory spectrum on Gram-positive bacteria. Sensitivity of Gram-negative bacteria increases in combination with outer-membrane permeabilizing treatments. Eukaryotic cells are bacteriocin-resistant. This cationic peptide inserts into bacterial membranes and causes membrane permeabilization, leading ultimately to cell death. Microarray analysis revealed sets of up-regulated and down-regulated genes in Bacillus cereus cells treated with sublethal bacteriocin concentration. Enterocin AS-48 can be purified in two steps or prepared as lyophilized powder from cultures in whey-based substrates. The potential applications of enterocin AS-48 as a food biopreservative have been corroborated against foodborne pathogens and/or toxigenic bacteria (Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enterica) and spoilage bacteria (Alicyclobacillus acidoterrestris, Bacillus spp., Paenibacillus spp., Geobacillus stearothermophilus, Brochothrix thermosphacta, Staphylococcus carnosus, Lactobacillus sakei and other spoilage lactic acid bacteria). The efficacy of enterocin AS-48 in food systems increases greatly in combination with chemical preservatives, essential oils, phenolic compounds, and physico-chemical treatments such as sublethal heat, high-intensity pulsed-electric fields or high hydrostatic pressure.
PMCID: PMC4284732  PMID: 25493478
enterocin; circular bacteriocin; antibacterial; membrane; food preservation

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