Friday, July 27, 2007

Rich pickings for proposals in dairy microbe biotechnology

This is what a quick literature search with PubMed can reveal:

J Bacteriol. 2007 Apr;189(8):3256-70. Epub 2007 Feb 16.
Complete genome sequence of the prototype lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363.
Wegmann U, O'Connell-Motherway M, Zomer A, Buist G, Shearman C, Canchaya C, Ventura M, Goesmann A, Gasson MJ, Kuipers OP, van Sinderen D, Kok J.
Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.

Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category "carbohydrate metabolism and transport," by far the largest category of novel proteins in
comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the "lateral gene transfer hot spot" in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.
PMID 17307855 [PubMed - indexed for MEDLINE]

Mol Microbiol. 1998 Aug;29(4):1029-38.
Comment in:
Mol Microbiol. 1999 Mar;31(5):1598-600.
Sequence and analysis of the 60 kb conjugative, bacteriocin-producing plasmid pMRC01 from Lactococcus lactis DPC3147.
Dougherty BA, Hill C, Weidman JF, Richardson DR, Venter JC, Ross RP. The Institute for Genomic Research, Rockville, MD, USA.

The complete sequence of pMRC01, a large conjugative plasmid from Lactococcus lactis ssp. lactis DPC3147, has been determined. Using a shotgun sequencing approach, the 60,232 bp plasmid sequence was obtained by the assembly of 1056 underlying sequences (sevenfold average redundancy). Sixty-four open reading frames (ORFs) were identified. Analysis of the gene organization of pMRC01 suggests that the plasmid can be divided into three functional domains, with each approximately 20 kb region separated by insertion sequence (IS) elements. The three regions are (i) the conjugative transfer region, including a 16-gene Tra (transfer) operon; (ii) the bacteriocin production region, including an operon responsible for the synthesis of the novel bacteriocin lacticin 3147; and (iii) the phage resistance and plasmid replication region of the plasmid. The complete sequence of pMRC01 provides important information about these industrially relevant phenotypes and gives insight into the structure, function and evolution of large gram-positive conjugative plasmids in general. The completely sequenced pMRC01 plasmid should also provide a useful framework for the design of novel
dairy industry.
PMID 9767571 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 2004 Jan;70(1):34-42.
Variable bacteriocin production in the commercial starter Lactococcus lactis DPC4275 is linked to the formation of the cointegrate plasmid pMRC02.
Trotter M, McAuliffe OE, Fitzgerald GF, Hill C, Ross RP, Coffey A.
Department of Microbiology, University College Cork, Ireland.

Lactococcus lactis DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with L. lactis MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin
3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage c2 infection of MG1363(pMRC02). While this locus is flanked by IS946 elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.
PMID 14711623 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 1996 Feb;62(2):612-9.
An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147.
Ryan MP, Rea MC, Hill C, Ross RP.
National Dairy Products Research Centre, Moorepark, Fermoy, County Cork, Republic
of Ireland.

Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition. The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci. On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147. The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L. lactis subsp. cremoris DPC4268. The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147.
The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials.
Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains. The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria. Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products.
PMID 8593062 [PubMed - indexed for MEDLINE]

J Appl Microbiol. 2003;95(6):1235-41.
A lacticin 481-producing adjunct culture increases starter lysis while inhibiting nonstarter lactic acid bacteria proliferation during Cheddar cheese ripening.
O'Sullivan L, Ross RP, Hill C.
Dairy Products Research Centre, Teagasc, Moorepark, Fermoy, County Cork, Ireland.

AIMS: The main aim of this study was to exploit a lacticin 481 producing strain, Lactococcus lactis CNRZ481, as an adjunct for Cheddar cheese manufacture, to increase starter cell lysis and control nonstarter lactic acid bacteria (NSLAB) proliferation in cheese. METHODS AND RESULTS: Lactococcus lactis CNRZ481 was exploited as an adjunct to L. lactis HP for the manufacture of Cheddar cheese at pilot scale (450 l). In these trials, inclusion of the adjunct strain did not compromise acid production by L. lactis HP and cheese was successfully manufactured within 5 h. Experimental cheese exhibited levels of lactate dehydrogenase (LDH) up to five-fold higher than control cheese and a significant reduction in NSLAB growth was also observed throughout the ripening period.
CONCLUSIONS: The aims of the study were accomplished as (i) greater enzyme release was achieved through lacticin 481-induced lysis which was associated with an improved flavoured cheese as assessed by a commercial grader and (ii) NSLAB growth was controlled, thus reducing the risk of off-flavour development.
SIGNIFICANCE AND IMPACT OF THE STUDY: The use of lacticin 481-producing adjuncts for cheese manufacture may prove beneficial for manufacturers who aim to achieve faster ripening through premature and elevated intracellular enzyme release while minimizing inconsistencies in cheese quality because of NSLAB activity.
PMID 14632996 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 2001 Jun;67(6):2699-704.
Strategy for manipulation of cheese flora using combinations of lacticin 3147-producing and -resistant cultures.
Ryan MP, Ross RP, Hill C.
Dairy Products Research Centre, Fermoy, County Cork, Ireland.

The aim of the present study was to develop adjunct strains which can grow in the presence of bacteriocin produced by lacticin 3147-producing starters in fermented products such as cheese. A Lactobacillus paracasei subsp. paracasei strain (DPC5336) was isolated from a well-flavored, commercial cheddar cheese and exposed to increasing concentrations (up to 4,100 arbitrary units [AU]/ml) of lantibiotic lacticin 3147. This approach generated a stable, more-resistant variant of the isolate (DPC5337), which was 32 times less sensitive to lacticin 3147 than DPC5336. The performance of DPC5336 was compared to that of DPC5337 as adjunct cultures in two separate trials using either Lactococcus lactis DPC3147 (a natural producer) or L. lactis DPC4275 (a lacticin 3147-producing transconjugant) as the starter. These lacticin 3147-producing starters were previously shown to control adventitious nonstarter lactic acid bacteria in cheddar cheese. Lacticin 3147 was produced and remained stable during ripening,
with levels of either 1,280 or 640 AU/g detected after 6 months of ripening. The more-resistant adjunct culture survived and grew in the presence of the bacteriocin in each trial, reaching levels of 10(7) CFU/g during ripening, in contrast to the sensitive strain, which was present at levels 100- to 1,000-fold lower. Furthermore, randomly amplified polymorphic DNA-PCR was employed to demonstrate that the resistant adjunct strain comprised the dominant microflora
in the test cheeses during ripening.
PMID 11375183 [PubMed - indexed for MEDLINE]

J Food Prot. 2001 Jan;64(1):81-6.
The effects of cultivating lactic starter cultures with bacteriocin-producing lactic acid bacteria.
Oumer A, Garde S, Gaya P, Medina M, Nuñez M.
Departamento de Tecnologiá de Alimentos, INIA, Madrid, Spain.

The effects of bacteriocins produced by six strains of lactic acid bacteria on 9 mesophilic and 11 thermophilic commercial starter cultures were investigated in mixed cultures of commercial starters with bacteriocin-producing strains in milk.
The bacteriocins produced by the test organisms were nisin A, nisin Z, lacticin 481, enterocin AS-48, a novel enterocin, and a novel plantaricin. Mesophilic commercial starters were in most cases tolerant of bacteriocins, with only two of the starters being partially inhibited, one by four and the other by two bacteriocins. The aminopeptidase activities of mesophilic starters were generally low, and only one of the combinations of mesophilic starter-bacteriocin producer gave double the aminopeptidase activity of the starter culture without the bacteriocin producer. Thermophilic commercial starters were more sensitive to bacteriocins than mesophilic starters, with six thermophilic starters being partially inhibited by at least one of the bacteriocins. Their aminopeptidase activities were generally higher than those of the mesophilic starters. The aminopeptidase activities of seven thermophilic starters were increased in the presence of bacteriocins, by factors of up to 9.0 as compared with the corresponding starter cultures alone. Bacteriocin-producing strains may be used as adjunct cultures to mesophilic starters for the inhibition of pathogens in soft and semihard cheeses, because mesophilic starters are rather tolerant of bacteriocins. Bacteriocin producers may also be used as adjunct cultures to thermophilic starters of high aminopeptidase activity, more sensitive to lysis by bacteriocins than mesophilic starters, for the acceleration of ripening in semihard and hard cheeses.
PMID 11198445 [PubMed - indexed for MEDLINE]

J Food Prot. 2005 May;68(5):1026-33.
Effect of milk inoculation with bacteriocin-producing lactic acid bacteria on a Lactobacillus helveticus adjunct cheese culture.
Avila M, Garde S, Medina M, Nuñez M.
Departamento de Tecnología de Alimentos, INIA, Carretera de La Coruña Km 7,
Madrid, 28040 Spain.

The effect of eight strains of lactic acid bacteria (two strains of Enterococcus, one strain of Lactobacillus, and five strains of Lactococcus, which produce enterocin AS-48, enterocin 607, nisin A, nisin Z, plantaricin 684, lacticin 481, or nisin Z plus lacticin 481) on acid production and proteolytic activity of Lactobacillus helveticus LH 92 (a highly peptidolytic strain used as an adjunct in cheese making) was evaluated in mixed cultures in milk. Acid production by mixed cultures depended on the sensitivity of L. helveticus LH 92 to the different bacteriocins and on the acidification rates of bacteriocin-producing strains. Proteolysis values of mixed cultures were, in all cases, lower than those of L. helveticus LH 92 single culture (control). Cell-free aminopeptidase activity values after 9 h of incubation did not increase in the presence of enterocin producers or the nisin A producer, whereas in the presence of the nisin Z producer, cell-free aminopeptidase activity was, at most, 3.7-fold greater than the control value. In mixed cultures with the plantaricin producer, a progressive lysis of L. helveticus LH 92 took place, with cell-free aminopeptidase activity values after 9 h being, at most, 10.5-fold greater than the control value. The highest cell-free aminopeptidase activity values after 9 h were recorded in the presence of lacticin 481 producers, with the values being, at most, 25.1-fold greater than the control value. L. helveticus LH 92 was extremely sensitive to small variations in the concentration of the inoculum of the nisin Z plus lacticin 481 producer, with there being a narrow optimum for the release of intracellular aminopeptidases. Plantaricin and lacticin 481 producers seemed the most promising strains to be combined with L. helveticus LH 92 as lactic cultures
for cheese manufacture,because of the accelerated release of intracellular
PMID 15895737 [PubMed - indexed for MEDLINE]

Int J Food Microbiol. 2006 Dec 1;112(3):230-5. Epub 2006 Jun 9.
Potential of lactic acid bacteria isolated from specific natural niches in food production and preservation.
Topisirovic L, Kojic M, Fira D, Golic N, Strahinic I, Lozo J.
Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a,
11010 Belgrade, Serbia and Montenegro. lab6@eunet.yu

Autochthonous strains of lactic acid bacteria (LAB) have been isolated from traditionally homemade cheeses collected from specific ecological localities across Serbia and Montenegro. Genetic and biochemical analysis of this LAB revealed that they produce bacteriocins, proteinases and exopolysaccharides. LAB produces a variety of antimicrobial substances with potential importance for food fermentation and preservation. Apart from the metabolic end products, some strains also secrete antimicrobial substances known as bacteriocins. Among the
natural isolates of LAB from homemade cheeses, bacteriocin producers were found in both lactococci and lactobacilli. Lactococcus lactis subsp. lactis BGMN1-5 was found to produce three narrow spectrum class II heat-stable bacteriocins. In addition to bacteriocin production, BGMN1-5 synthesized a cell envelope-associated proteinase (CEP) and shows an aggregation phenotype. Another isolate, L. lactis subsp. lactis BGSM1-19 produces low molecular mass (7 kDa) bacteriocin SM19 that showed antimicrobial activity against Staphylococcus aureus, Micrococcus flavus and partially against Salmonella paratyphi. Production of bacteriocin reaches a plateau after 8 h of BGSM1-19 growth. Bacteriocin SM19 retained activity within the wide pH range from 1 to 12 and after the treatment at 100 degrees C for 15 min. Among collection of lactobacilli, the isolate Lactobacillus paracasei subsp. paracasei BGSJ2-8 produces heat-stable bacteriocin SJ (approx. 5 kDa) polypeptide. It retained activity after treatment for 1 h at 100 degrees C, and in the pH range from 2 to 11. In addition to isolates from cheeses, bacteriocin-producing human oral lactobacilli were detected. Most of them showed antimicrobial activity against streptococci, staphylococci and micrococci, but not against Candida. Isolate BGHO1 that showed the highest antimicrobial activity was determined as Interestingly, L. paracasei. Lactobacillus helveticus BGRA43, which was isolated from the human intestine showed strong activity against Clostridium sporogenes, but it was not possible to detect any bacteriocin production in this isolate by using standard procedures.
Further analysis of antimicrobial activity revealed that BGRA43 has a relatively broad spectrum. Lactobacilli resistant to nisin were also detected among natural isolates. They produce bacteriocins, which have no activity against nisin producing lactococci.
PMID 16764959 [PubMed - indexed for MEDLINE]

Int J Food Microbiol. 1999 Dec 15;53(2-3):141-52.
Occurrence of nisin Z production in Lactococcus lactis BFE 1500 isolated from wara, a traditional Nigerian cheese product.
Olasupo NA, Schillinger U, Narbad A, Dodd H, Holzapfel WH.
Department of Botany and Microbiology, Faculty of Science, Lagos State University
Ojo, Nigeria.

Screening for bacteriocin production of 500 strains of lactic acid bacteria (LAB) from various African fermented foods resulted in the detection of a bacteriocin producing Lactococcus lactis (BFE 1500) isolated from a dairy product called wara. The bacteriocin inhibited not only the closely related LAB, but also strains of Listeria monocytogenes, Listeria innocua, Clostridium butyricum, Clostridium perfringens, Bacillis cereus and Staphylococcus aureus. It was heat
stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10), but highest activity was observed in the lower pH range. The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K, but not by other proteases. Growth kinetic assay indicated stronger growth inhibition by the bacteriocin produced by Lc. lactis BFE 1500 on L. monocytogenes WS 2250 and B. cereus DSM 2301 than with the nisin A producing strain DSM 20729.
Polymerase chain reaction indicated the presence of the nisin operon in strain BFE 1500 and sequencing of its structural gene showed that Lc. lactis BFE 1500 produced the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The genetic determinants for bacteriocin production in strain BFE 1500 are located on a conjugative transposon. The ability of the bacteriocin produced by Lc. lactis BFE 1500 to
inhibit a wide range of food-borne pathogens is of special interest for food safety, especially in the African environment with perennial problems of poor food hygiene.
PMID 10634705 [PubMed - indexed for MEDLINE]

Int J Food Microbiol. 2003 Mar 15;81(2):137-45.
Isolation of nisin-producing Lactococcus lactis WNC 20 strain from nham, a traditional Thai fermented sausage.
Noonpakdee W, Santivarangkna C, Jumriangrit P, Sonomoto K, Panyim S.
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok
10400, Thailand.

A total of 14,020 lactic acid bacteria (LAB) were isolated from nham and screened for bacteriocin production. One Lactococcus lactis strain WNC 20 produced a bacteriocin that not only inhibited closely related LAB, but also some food-borne pathogens including Listeria monocytogenes, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus. Biochemical studies revealed that the bacteriocin was heat-stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10). The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K but not other proteases. The antimicrobial spectrum and some characteristics of this bacteriocin were nearly identical to
that of nisin. The gene encoding this bacteriocin was amplified by polymerase chain reaction (PCR) with nisin gene-specific primer. Sequencing of this gene showed identical sequences to nisin Z as indicated by the substitution of asparagine residue instead of histidine at position 27. The ability of the bacteriocin produced by Lc. lactis WNC 20 may be useful in improving the food safety of the fermented product.
PMID 12457588 [PubMed - indexed for MEDLINE]

J Basic Microbiol. 1997;37(3):187-96.
Production of nisin-like bacteriocins by Lactococcus lactis strains isolated from vegetables.
Franz CM, Du Toit M, von Holy A, Schillinger U, Holzapfel WH.
Bundesforschungsanstalt für Ernährung, Institut für Hygiene and Toxikologie,
Karlsruhe, Germany.

Four bacteriocin producing lactic acid bacteria isolated from vegetables were identified as Lactococcus lactis strains on the basis of physiological and biochemical characteristics, carbohydrate fermentation patterns and analysis of total soluble protein pattern by SDS PAGE. The bacteriocins had a wide spectrum of activity as antagonism was detected not only towards a variety of lactic acid bacteria, but also to Staphylococcus aureus and Listeria monocytogenes. These bacteriocins were resistant to heating at 121 degree C for 15 minutes and showed highest activity at low pH (less than 5.0). They were inactivated by the proteolytic enzymes alpha-chymotrypsin and proteinase K, but not by lipase, alpha-amylase, catalase or lysozyme. These bacteriocinogenic Lactococcus strains were all immune to the bacteriocins produced as well as to commercial nisin. Bacteriocin producer culture supernatants showed a high degree (70 or 100%) of cross-reactivity in the nisin ELISA, suggesting similarity of the produced bacteriocins to nisin. The potential application of bacteriocin producing lactococci of vegetable origin for safety assurance of vegetable foods and controlling vegetable fermentations is
PMID 9265741 [PubMed - indexed for MEDLINE]

Int J Food Microbiol. 1992 Jun;16(2):141-51.
Identification and characterization of two bacteriocin-producing strains of Lactococcus lactis isolated from vegetables.
Uhlman L, Schillinger U, Rupnow JR, Holzapfel WH.
Federal Research Centre for Nutrition, Institute of Hygiene and Toxicology, Karlsruhe, Germany.

Isolated from mixed salad and fermented carrots, 123 strains of lactic acid bacteria were screened for bacteriocin production. Two strains, D53 and 23, identified as Lactococcus lactis by DNA-DNA hybridizations, produced heat stable bacteriocins which were resistant to trypsin and pepsin, but were inactivated by alpha-chymotrypsin and proteinase K. The bacteriocins were active from pH 2 to 9 and inhibited species of Listeria, Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Carnobacterium, Bacillus and Staphylococcus. Strain D53 produced bacteriocin at pH values of 4.5-8.0 and from 10 to 37 degrees C.
PMID 1445757 [PubMed - indexed for MEDLINE]

J Appl Microbiol. 2000 Apr;88(4):563-71.
Production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from Kimchi.
Choi HJ, Cheigh CI, Kim SB, Pyun YR.
Department of Biotechnology and Bioproducts Research Center, Yonsei University,
Seoul, Korea.

Lactococcus lactis subsp. lactis A164 was isolated from Kimchi (Korean traditional fermented vegetables). The bacteriocin produced by strain A164 was active against closely related lactic acid bacteria and some food-borne pathogens including Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. The antimicrobial spectrum was nearly identical to that of nisin.
Bacteriocin activity was not destroyed by exposure to elevated temperatures at low pH values, but the activity was lost at high pH values. This bacteriocin was inactivated by pronase E and alpha, beta-chymotrypsin, but not by trypsin, pepsin, and alpha-amylase. Cultures of L. lactis subsp. lactis A164 maintained at a constant pH of 6.0 exhibited maximum production of the bacteriocin. It was purified to homogeneity by ammonium sulphate precipitation, sequential ion
exchange chromatography, and ultrafiltration. Tricine-SDS-PAGE of purified bacteriocin gave the same molecular weight of 3.5 kDa as that of nisin. The gene encoding this bacteriocin was amplified by PCR with nisin gene-specific primers and sequenced. It showed identical sequences to the nisin gene. These results indicate that bacteriocin produced by Lactococcus lactis A164 is a nisin-like bacteriocin.
PMID 10792514 [PubMed - indexed for MEDLINE]

Curr Microbiol. 2003 May;46(5):385-8.
Identification and characteristics of nisin Z-producing Lactococcus lactis subsp.
lactis isolated from Kimchi.
Park SH, Itoh K, Kikuchi E, Niwa H, Fujisawa T.
Laboratory of Veterinary Public Health, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

We isolated bacteriocin-producing Lactococcus lactis subsp. lactis from Kimchi.
The bacteriocin inhibited strains of Clostridium perfringens, C. difficile, Listeria monocytogenes, vancomycin-resistant Enterococcus, and one out of four methicillin-resistant Staphylococcus aureus strains, as well as some closely related lactic acid bacteria. In tricine-SDS-PAGE, the bacteriocin migrated with an apparent molecular weight of about 4 kDa to the same location as nisin A and crude nisin Z. The gene encoding this bacteriocin was found to be identical to that of nisin Z with direct PCR sequence methods. The inhibitory activity was
stable against heat and pH, but it was lost at 100 degrees C for 1 h and at 121 degrees C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or beta-glucosidase. There were vsome differences in characteristics from those of nisins described previously.
PMID 12732968 [PubMed - indexed for MEDLINE]

J Appl Microbiol. 2004;97(3):621-8.
Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese.
Ghrairi T, Manai M, Berjeaud JM, Frère J.
Laboratoire de Biochimie et Biologie Moléculaire, Faculté des Sciences de Tunis,
Campus universitaire, Tunis, Tunisia.

AIMS: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great significance for the dairy industry to improve food safety. METHODS AND RESULTS: Six-hundred strains of LAB isolated from 'rigouta', a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identified as Lactococcus lactis and produced nisin Z as
demonstrated by mass spectrometry analysis of the purified antibacterial compound. Polymerase chain reaction experiments using nisin gene-specific primers confirmed the presence of nisin operon. Plasmid profiles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identified, at molecular level, as Enterococcus faecalis. The purified bacteriocin produced by this strain showed a molecular mass of 10 201.33 +/- 0.85 Da. This new member of class III
bacteriocins was termed enterocin MMT05. CONCLUSIONS: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or beneficial role in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety. Copyright 2004 The Society for Applied Microbiology
PMID 15281944 [PubMed - indexed for MEDLINE]

Int J Food Microbiol. 2006 Mar 15;107(2):138-47. Epub 2005 Nov 8.
Streptococcus macedonicus ACA-DC 198 produces the lantibiotic, macedocin, at temperature and pH conditions that prevail during cheese manufacture.
Van den Berghe E, Skourtas G, Tsakalidou E, De Vuyst L.
Research Group of Industrial Microbiology, Fermentation Technology and Downstream
Processing (IMDO), Department of Applied Biological Sciences, Vrije Universiteit
Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium.

Streptococcus macedonicus ACA-DC 198, a natural cheese isolate, produces the anticlostridial bacteriocin, macedocin. Bacteriocin activity was detected from the mid-exponential growth phase and remained constant during the stationary phase. A secondary model was setup to describe the influence of temperature (20-45 degrees C) and pH (5.1-6.9) on cell growth of and bacteriocin production by S. macedonicus ACA-DC 198 during in vitro laboratory fermentations. The optimum temperature for bacteriocin production (20-25 degrees C) was markedly lower than the optimum growth temperature (42.3 degrees C). In contrast, the
specific macedocin production was maximal around pH 6.0, whereas growth was optimal at pH 6.4. Consequently, the maximum bacteriocin activity was reached between pH 6.0 and 6.5.
PMID 16288813 [PubMed - indexed for MEDLINE]

J Food Prot. 2004 Dec;67(12):2727-34.
Characterization and antimicrobial activity of bacteriocin 217 produced by natural isolate Lactobacillus paracasei subsp. paracasei BGBUK2-16.
Lozo J, Vukasinovic M, Strahinic I, Topisirovic L.
Institute of Molecular Genetics and Genetic Engineering, 11010 Belgrade, Serbia
and Montenegro.

The strain Lactobacillus paracasei subsp. paracasei BGBUK2-16. which was isolated from traditionally homemade white-pickled cheese, produces bacteriocin 217 (Bac217; approximately 7 kDa). The onset of Bac217 biosynthesis was observed in the logarithmic phase of growth, and the production plateau was reached after 9 or 12 h of incubation at 37 and 30 degrees C, respectively, when culture entered the early stationary phase. Biochemical characterization showed that Bac217 retained antimicrobial activity within the range of pH 3 to 12 or after treatment at 100 degrees C for 15 min. Bac217 antimicrobial activity also remained unchanged after storage at 4 degrees C for 6 months or -20 degrees C for up to 12 months. However, Bac217 activity was completely lost after treatment with different proteolytic enzymes. BGBUK2-16 contains only one plasmid about 80 kb in size. Plasmid curing indicated that genes coding for Bac217 synthesis and immunity seem to be located on this plasmid. Bac217 exhibited antimicrobial activity against some pathogenic bacteria, such as Staphylococcus aureus and
Bacillus cereus. Interestingly, Bac217 showed activity against Salmonella sp. and Pseudomonas aeruginosa ATCC27853. The inhibitory effect of BGBUK2-16 on the growth of S. aureus in mixed culture was observed. S. aureus treatment with Bac217 led to a considerable decrease (CFU/ml) within a short period of time. The mode of Bac217 action on S. aureus was identified as bactericidal. It should be noted that the strain BGBUK2-16 was shown to be resistant to bacteriocin nisin, which is otherwise widely used as a food additive for fermented dairy products.
PMID 15633678 [PubMed - indexed for MEDLINE]

J Dairy Sci. 2006 Aug;89(8):2882-93.
Effect of high-pressure treatment and a bacteriocin-producing lactic culture on the proteolysis, texture, and taste of Hispánico cheese.
Avila M, Garde S, Gaya P, Medina M, Nuñez M.
Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y
Tecnología Agraria y Alimentaria (INIA) Madrid, 28040 Spain.

The effects of high-pressure treatment, by itself or in combination with a bacteriocin-producing culture added to milk, on the proteolysis, texture, and taste of Hispánico cheese were investigated. Two vats of cheese were manufactured from a mixture of cow and ewe milk. Milk in one vat was inoculated with 0.5% Lactococcus lactis ssp. lactis INIA 415, a nisin Z and lacticin 481 producer; 0.5% L. lactis ssp. lactis INIA 415-2, a bacteriocin-nonproducing mutant; and 2% of a commercial Streptococcus thermophilus culture. Milk in the other vat was inoculated with 1% L. lactis ssp. lactis INIA 415-2 and 2% S. thermophilus culture. After ripening for 15 d at 12 degrees C, half of the cheeses from each vat were treated at 400 MPa for 5 min at 10 degrees C. Ripening of high-pressure-treated and untreated cheeses continued at 12 degrees C until d 50.
High-pressure treatment of cheese made from milk without the bacteriocin producer accelerated casein degradation and increased the free AA content, but it did not significantly influence the taste quality or taste intensity of the cheese.
Addition of the bacteriocin producer to milk lowered the ratio of hydrophobic peptides to hydrophilic peptides, increased the free AA content, and enhanced the taste intensity. The combination of milk inoculation with the bacteriocin producer and high-pressure treatment of the cheese resulted in higher levels of both hydrophobic and hydrophilic peptides but had no significant effect on the free AA content, taste quality, or taste intensity.
PMID 16840604 [PubMed - indexed for MEDLINE]

J Appl Microbiol. 2006;100(1):135-43.
Evaluation of live-culture-producing lacticin 3147 as a treatment for the control of Listeria monocytogenes on the surface of smear-ripened cheese.
O'Sullivan L, O'connor EB, Ross RP, Hill C.
Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland.

AIMS: A live Lactococcus lactis culture, producing the two-component broad spectrum bacteriocin lacticin 3147, was assessed for ability to inhibit the food pathogen Listeria monocytogenes on the surface of smear-ripened cheese. METHODS AND RESULTS: In initial experiments, the addition of Listeria to a lacticin 3147-containing fermentate produced with L. lactis DPC4275 (a transconjugant strain derived from L. lactis DPC3147) resulted in at least a 4 log reduction of the pathogen in 30 min. Two separate trials were performed in order to assess the most suitable method for application of the potential protective culture to smear-ripened cheese. In the initial trial, the L. lactis was sprayed onto the surface of the cheese either before or after Listeria was deliberately applied.
Application of the culture following Listeria challenge, yielded up to a 1000-fold reduction of the pathogen in contrast to the pretreatment where Listeria numbers were unaffected. In a further trial, three applications of the live lacticin 3147-producing culture was used on a cheese surface containing Listeria. Listeria numbers were found to be up to 100-fold lower than in the cheese treated with L. lactis DPC4268 (control). CONCLUSION: While application of the live lacticin 3147 producer did not give complete elimination of the pathogen the results nonetheless demonstrate the potential of the bioprotectant for improving the safety of smear-ripened cheeses and particularly those that contain low level contamination with Listeria. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of lacticin 3147 as a live-culture can serve as a bioprotectant for the control of L. monocytogenes on the surface of smear-ripened cheese.
PMID 16405693 [PubMed - indexed for MEDLINE]

FEMS Microbiol Lett. 1993 Sep 15;112(3):313-8.
Conjugal transfer of the determinants for bacteriocin (lacticin 481) production and immunity in Lactococcus lactis subsp. lactis CNRZ 481.
Piard JC, Delorme C, Novel M, Desmazeaud M, Novel G.
INRA, Station de Recherches Laitières, Jouy-en-Josas, France.

The lacticin 481-producer (Lct+), L. lactis subsp. lactis (L. lactis) CNRZ 481 harbours 5 plasmids of 6.5, 7.5, 20, 37 and 69 kb. Novobiocin treatment of L. lactis 481 led to the appearance of lacticin 481 deficient variants which had all lost the 69 kb plasmid. Conjugal transfer of the lacticin 481 structural gene (lct) into the plasmid free strain L. lactis IL1441 yielded Lct+ transconjugants at a 10(-4) frequency, which carried a plasmid with an apparent size of 120-130
kb. Southern hybridization analyses showed that the lct gene was located on the 69 kb plasmid in L. lactis 481 and on the 120-130 kb plasmid in the transconjugants. The lct gene was in higher copy number in transconjugants than in the parental strain resulting in two-fold higher lacticin 481 production in the former strain.
PMID 8224796 [PubMed - indexed for MEDLINE]

Mol Nutr Food Res. 2006 Mar;50(3):306-13.
Characterization of bacteriocins from two Lactococcus lactis subsp. lactis isolates.
Akçelik O, Tükel C, Ozcengiz G, Akçelik M.
Department of Biotechnology, Middle East Technical University, Ankara, Turkey.

In this study, bacteriocins from two Lactococcus lactis subsp. lactis isolates from raw milk samples in Turkey designated OC1 and OC2, respectively, were characterized and identified. The activity spectra of the bacteriocins were determined by using different indicator bacteria including Listeria, Bacillus and Staphylococcus spp. Bacteriocins were tested for their sensitivity to different enzymes, heat treatments and pH values. Loss of bacteriocin activities after alpha-amylase treatment suggested that they form aggregates with carbohydrates.
Molecular masses of the purified bacteriocins were determined by SDS-PAGE. PCR amplification was carried out with specific primers for the detection of their structural genes. As a result of these studies, the two bacteriocins were characterized as nisin and lacticin 481, respectively. Examination of plasmid contents of the isolates and the results of plasmid curing and conjugation experiments showed that in L. lactis subsp. lactis OC1 strain the 39.7-kb plasmid is responsible for nisin production, lactose fermentation and proteolytic activity, whereas the 16.0-kb plasmid is responsible for lacticin 481 production and lactose fermentation in L. lactis subsp. lactis OC2 strain.
PMID 16523441 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 1997 Apr;63(4):1434-40.
Application and evaluation of the phage resistance- and bacteriocin-encoding plasmid pMRC01 for the improvement of dairy starter cultures.
Coakley M, Fitzgerald G, Ros RP.
National Dairy Products Research Centre, Fermoy, County Cork, Ireland.

The conjugative 63-kb lactococcal plasmid pMRC01 encodes bacteriophage resistance and production of and immunity to a novel broad-spectrum bacteriocin, designated lacticin 3147 (M.P. Ryan, M.C. Rea, C. Hill, and R.P. Ross, Appl. Environ. Microbiol. 62:612-619, 1996). The phage resistance is an abortive infection mechanism which targets the phage-lytic cycle at a point after phage DNA replication. By using the genetic determinants for bacteriocin immunity encoded on the plasmid as a selectable marker, pMRC01 was transferred into a variety of lactococcal starter cultures to improve their phage resistance properties.
Selection of resulting transconjugants was performed directly on solid media containing the bacteriocin. Since the starters exhibited no spontaneous resistance to the bacteriocin as a selective agent, this allowed the assessment of the transfer of the naturally occurring plasmid into a range of dairy starter cultures. Results demonstrate that efficient transfer of the plasmid was dependent on the particular recipient strain chosen, and while high-frequency transfer (10(-3) per donor) of the entire plasmid to some strains was observed, the plasmid could not be conjugated into a number of starters. In this study, transconjugants for a number of lactococcal starter cultures which are phage resistant and bacteriocin producing have been generated. This
bacteriocin-producing phenotype allows for control of nonstarter flora in food fermentations, and the phage resistance property protects the starter cultures in industry. The 63-kb plasmid was also successfully transferred into Lactococcus lactis MG1614 cells via electroporation.
PMID 9097441 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 2001 Jun;67(6):2853-8.
Exploitation of plasmid pMRC01 to direct transfer of mobilizable plasmids into commercial lactococcal starter strains.
Hickey RM, Twomey DP, Ross RP, Hill C.
Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork.

Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (oriT). This oriT-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 x 10(-4)
transconjugants/donor cell from an MG1363 (recA mutant) host containing pMRC01.
All of the resultant chloramphenicol-resistant transconjugants contained both pRH001 and genetic determinants responsible for bacteriocin production and immunity of pMRC01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pMRC01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the oriT-containing fragment was exploited to mobilize pRH001 alone to a number of lactococcal hosts. These results demonstrate that oriT of pMRC01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform.
PMID 11375207 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 2001 Feb;67(2):929-37.
Naturally occurring lactococcal plasmid pAH90 links bacteriophage resistance and mobility functions to a food-grade selectable marker.
O' Sullivan D, Ross RP, Twomey DP, Fitzgerald GF, Hill C, Coffey A.
Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Ireland.

The bacteriophage resistance plasmid pAH90 (26,490 bp) is a natural cointegrate plasmid formed via homologous recombination between the type I restriction-modification specificity determinants (hsdS) of two smaller lactococcal plasmids, pAH33 (6,159 bp) and pAH82 (20,331 bp), giving rise to a bacteriophage-insensitive mutant following phage challenge (D. O'Sullivan, D. P.
Twomey, A. Coffey, C. Hill, G. F. Fitzgerald, and R. P. Ross, Mol. Microbiol.
36:866-876; 2000). In this communication we provide evidence that the recombination event is favored by phage infection. The entire nucleotide sequence of plasmid pAH90 was determined and found to contain 24 open reading frames (ORFs) responsible for phenotypes which include restriction-modification, phage adsorption inhibition, plasmid replication, cadmium resistance, cobalt transport, and conjugative mobilization. The cadmium resistance property, encoded by the cadA gene, which has an associated regulatory gene (cadC), is of particular interest, as it facilitated the selection of pAH90 in other phage-sensitive lactococci after electroporation. In addition, we report the identification of a group II self-splicing intron bounded by two exons which have the capacity to encode a relaxase implicated in conjugation in gram-positive bacteria. The functionality of this intron was evident by demonstrating splicing in vivo. Given that pAH90 encodes potent phage defense systems which act at different stages in the phage lytic cycle, the linkage of these with a food-grade selectable marker on a replicon that can be mobilized among lactococci has significant potential for natural strain improvement for industrial dairy fermentations which are susceptible to phage inhibition.
PMID 11157264 [PubMed - indexed for MEDLINE]

J Dairy Sci. 2001 Jul;84(7):1610-20.
DNA sequence analysis of three Lactococcus lactis plasmids encoding phage resistance mechanisms.
Boucher I, Emond E, Parrot M, Moineau S.
Department of Biochemistry and Microbiology, Faculté des Sciences et de Génie,
Faculté de Médecine Dentaire, Université Laval, Quebec, Canada.

The three Lactococcus lactis plasmids pSRQ700, pSRQ800, and pSRQ900 encode the previously described anti-phage resistance mechanisms LlaDCHI, AbiK, and AbiQ, respectively. Since these plasmids are likely to be introduced into industrial Lactococcus lactis strains used to manufacture commercial fermented dairy products, their complete DNA sequences were determined and analyzed. The plasmids pSRQ700 (7784 bp), pSRQ800 (7858 bp), and pSRQ900 (10,836 bp) showed a similar genetic organization including a common lactococcal theta-type replicon. A second replication module showing features of the pMV158 family of rolling circle replicons was also found on pSRQ700. The theta replication regions of the three
plasmids were associated with two additional coding regions, one of which encodes for HsdS, the specificity subunit of the type I restriction/modification system.
When introduced into L. lactis IL1403, the HsdS of pSRQ800 and pSRQ900 conferred a weak resistance against phage P008 (936 species). These results indicated that both HsdS subunits can complement the chromosomally encoded type I restriction/modification system in IL1403. The genes involved in the phage resistance systems LlaDCHI, AbiK, and AbiQ were found in close proximity to and downstream of the replication modules. In pSRQ800 and pSRQ900, transfer origins and putative tyrosine recombinases were found upstream of the theta replicons.
Genes encoding recombination proteins were also found on pSRQ700. Finally, open reading frames associated with bacteriocin production were found on pSRQ900, but no anti-lactococcal activity was detected. Based on our current knowledge, these three plasmids are safe and suitable for food-grade applications.
PMID 11467810 [PubMed - indexed for MEDLINE]

Microbiology. 1994 Jun;140 ( Pt 6):1291-300.
The majority of lactococcal plasmids carry a highly related replicon.
Seegers JF, Bron S, Franke CM, Venema G, Kiewiet R.
Department of Genetics, Centre of Biological Sciences, Haren, The Netherlands.

DNA sequence analysis and Southern hybridizations, together with complementation experiments, were used to study relationships between lactococcal plasmid replicons. pWVO2, pWVO4 and pWVO5, which co-exist in Lactococcus lactis subsp. cremoris Wg2, and pIL7 (isolated from another strain) all contained a functional replication region which appeared to be very similar to that of some known lactococcal plasmids. They contain a gene encoding a highly conserved RepB protein (60-80% amino acid identity between pWVO2, pWVO4 and pWVO5), which is essential for replication. When supplied in trans, repB of pWVO2 complemented a repB deficiency of pWVO5. Upstream of the repB gene, all these plasmids contain a strongly conserved region including a 22 bp sequence tandemly repeated three-and-a-half times, and an A/T-rich region. The similarity with pWVO2, which is known to replicate via a theta mechanism, suggests that all plasmids of this family are capable of theta replication. Southern hybridizations revealed that many lactococcal strains contain plasmids of this family.
PMID 8081493 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 2001 Apr;67(4):1700-9.
Molecular characterization of a theta replication plasmid and its use for development of a two-component food-grade cloning system for Lactococcus lactis.
Emond E, Lavallée R, Drolet G, Moineau S, LaPointe G.
Centre de recherche STELA, Département des sciences des aliments et de nutrition,
Université Laval, Québec, Canada G1K 7P4.

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing
erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile
of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.
PMID 11282624 [PubMed - indexed for MEDLINE]

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Anonymous modern farming methods said...

Dear Sir,
Thank you for this informative inputs.

November 02, 2012 5:30 pm  

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