Cloning and expression of a novel lactococcal aggregation factor from Lactococcus lactis subsp. lactis BGKP1

<p>Abstract</p> <p>Background</p> <p>Aggregation may play a main role in the adhesion of bacteria to the gastrointestinal epithelium and their colonization ability, as well as in probiotic effects through co-aggregation with intestinal pathogens and their subsequent removal. The aggregation phenomenon in lactococci is directly associated with the sex factor and lactose plasmid co-integration event or duplication of the cell wall spanning (CWS) domain of PrtP proteinase.</p> <p>Results</p> <p><it>Lactococcus lactis </it>subsp. <it>lactis </it>BGKP1 was isolated from artisanal semi-hard homemade cheese and selected due to its strong auto-aggregation phenotype. Subsequently, non-aggregating derivative (Agg^-) of BGKP1, designated as BGKP1-20, was isolated, too. Comparative analysis of cell surface proteins of BGKP1 and derivative BGKP1-20 revealed a protein of approximately 200 kDa only in the parental strain BGKP1. The gene involved in aggregation (<it>aggL</it>) was mapped on plasmid pKP1 (16.2 kb), cloned and expressed in homologous and heterologous lactococci and enterococci. This novel lactococcal aggregation protein was shown to be sufficient for cell aggregation in all tested hosts. In addition to the <it>aggL </it>gene, six more ORFs involved in replication (<it>repB </it>and <it>repX</it>), restriction and modification (<it>hsdS</it>), transposition (<it>tnp</it>) and possible interaction with mucin (<it>mbpL</it>) were also located on plasmid pKP1.</p> <p>Conclusion</p> <p>AggL is a new protein belonging to the collagen-binding superfamily of proteins and is sufficient for cell aggregation in lactococci.</p

Use of Lactobacillus helveticus BGRA43 for Manufacturing Fermented Milk Products

Soj bakterije Lactobacillus helveticus BGRA43, izoliran iz crijeva čovjeka, ima antimikrobni učinak na sojeve patogenih mikroorganizama u hrani, a tijekom fermentacije u mlijeku oslobađa peptide protuupalnih svojstava. U ovom je radu potvrđeno da soj BGRA43 djeluje antimikrobno na patogene sojeve, kao što su: Yersinia enterocolitica, Shigella sonnei, S. flexneri i Streptococcus pneumoniae. Stanice soja BGRA43 mogu preživjeti u simuliranim uvjetima gastrointestinalnog sustava uz dodatak mlijeka, pri čemu zadržavaju dovoljnu brojnost. Osim toga, LC/MS/MS analizom potvrđeno je da stanice soja BGRA43 mogu hidrolizirati β-laktoglobulin. U prisutnosti prebiotika, tj. inulina ili koncentrata β-glukana iz zobenih mekinja (Nutrim®) povećao se rast stanica soja BGRA43, čak i kad je prebiotik upotrijebljen kao jedini izvor ugljika. Soj BGRA43 odlično je rastao i u punomasnom kravljem ili kozjem mlijeku, te mlijeku koje je sadržavalo inulin ili Nutrim. Primjenom probiotičkog soja BGRA43 kao starter kulture dobiveni su fermentirani mliječni proizvodi od kravljeg ili kozjeg mlijeka sa ili bez dodatka inulina ili Nutrima s otprilike 107 CFU/mL stanica probiotika. Svi su proizvodi bili homogeni i viskozni, a najbolju su senzorsku ocijenu dobili fermentirani napici proizvedeni od rekonstituiranog mlijeka, punomasnog kravljeg mlijeka te kozjeg mlijeka s dodatkom 1 % inulina.Lactobacillus helveticus BGRA43 isolated from human intestines shows antimicrobial activity against foodborne pathogens and during fermentation in milk releases peptides with demonstrated anti-inflammatory properties. In this study, it was found that strain BGRA43 exhibits antimicrobial activity against human pathogens Yersinia enterocolitica, Shigella sonnei, S. flexneri and Streptococcus pneumoniae. Strain BGRA43 was able to survive in simulated gastric juice containing milk and retained cell number stability during the incubation in simulated intestinal conditions. In addition, LC/MS/MS analysis showed the ability of BGRA43 to hydrolyze β-lactoglobulin. Abundant growth of strain BGRA43 occurred in the presence of prebiotics inulin or concentrated oat bran β-glucan (Nutrim®), even when used as the sole carbon source. Similarly, strain BGRA43 grew satisfactorily in pure cow\u27s or goat\u27s milk as well as in the milk containing inulin or Nutrim®. Using the probiotic strain BGRA43 as a single starter strain, fermented milk products obtained from cow\u27s or goat\u27s milk with or without inulin or Nutrim® contained about 107 CFU/mL. The products were homogeneous and viscous and the best sensory scores were observed for fermented milk beverage made from reconstituted skimmed milk, whole cow\u27s milk and whole goat\u27s milk supplemented with 1 % inulin

Karakterizacija i odabir autohtonih bakterija mliječno-kiselog vrenja kao starter kultura u zanatskoj proizvodnji vlasinskog sira iz svježeg kozjeg mlijeka

The aim of this study is the isolation, characterization and identification of autochthonous lactic acid bacteria (LAB) from artisanal Vlasina raw goat\u27s milk cheese for the selection of potential starter cultures. Soft white Vlasina cheese was manufactured at a household on the Stara Planina Mountain using traditional techniques without starter cultures. One hundred and forty nine LAB isolates were collected from two samples of Vlasina cheese, designated as BGVL2 (5 days old) and BGVL2a (15 days old). The population of LAB in the cheese samples was characterized by phenotype-based assays and presumptively identified using repetitive element palindromic polymerase chain reaction (rep-PCR) with the primer (GTG)5. Results were confirmed by 16S rDNA sequencing. Among the BGVL2 isolates (56), the most numerous LAB species were Leuconostoc pseudomesenteroides (27) and Lactococcus lactis (26). In 15-day-old BGVL2a (93 isolates), Lactobacillus plantarum (33), Enterococcus durans (26) and Pediococcus pentosaceus (14) were predominant. Lc. lactis ssp. lactis BGVL2-8 showed good acidification ability and the ability to produce antimicrobial compounds, Lb. plantarum BGVL2a-18 had good proteolytic ability and produced exopolysaccharides, while BGVL2-29 and BGVL2-63, which belonged to the species Ln. pseudomesenteroides, utilized citrate and produced diacetyl and acetoin. They appeared to be suitable candidates for inclusion in the starter culture. This study contributed to the understanding of the role of autochthonous LAB in the quality of artisanal cheese and the possibility of using the selected LAB as potential starter cultures for cheese making under controlled conditions.Svrha je ovoga rada bila izolirati, karakterizirati i identificirati autohtone bakterije mliječno-kiselog vrenja koje se mogu upotrijebiti kao starter kulture u zanatskoj proizvodnji vlasinskog sira iz svježeg kozjeg mlijeka. Meki je bijeli vlasinski sir bio izrađen u domaćinstvu na Staroj Planini tradicionalnim postupkom bez dodatka starter kultura. Iz dvaju je uzoraka vlasinskih sireva, označenih kao BGVL2 (sir star 5 dana) i BGVL2 (sir star 15 dana) prikupljeno 149 izolata bakterija mliječno-kiselog vrenja. Populacija je bakterija iz uzoraka sireva okarakerizirana pomoću fenotipskih testova i primarno identificirana primjenom metode rep-PCR s početnicom (GTG)5. Rezultati su potvrđeni sekvenciranjem 16S rDNA. Među izolatima iz sira BGVL2 (njih 56) najbrojnije su bile bakterije Leuconostoc pseudomesenteroides (27) i Lactococcus lactis (26). U siru BGVL2a (93 izolata), starom 15 dana, prevladavale su vrste Lactobacillus plantarum (33), Enterococcus durans (26) i Pediococcus pentosaceus (14). Izolat Lc. lactis ssp. lactis BGVL2-8 pokazao je dobru sposobnost zakiseljavanja i proizvodnje antimikrobnih spojeva. Izolat Lb. plantarum BGVL2a-18 imao je dobru proteolitičku aktivnost i sposobnost proizvodnje egzopolisaharida, dok su izolati BGVL2-29 i BGVL2-63 vrste Ln. pseudomesenteroides za rast koristili citrate, a proizvodili diacetil i acetoin. Stoga su navedeni izolati odabrani kao starter kulture. Ovo je istraživanje pridonijelo razumijevanju uloge autohtonih bakterija mliječno-kiselog vrenja u zanatskoj proizvodnji kvalitetnog sira, te je potvrdilo mogućnost primjene odabranih izolata kao starter kultura pri izradi vlasinskog sira u kontroliranim uvjetima

PRINCIPLES OF LABELLING OF AUTOCHTONOUS CHEESES WITH GEOGRAPHICAL ORIGIN

Bearing in mind the increase of human population, development of new technologies and intensive care of human health, new challenges for scientists appeared. In the fields of fermented food based on various milks modern trends are going to production of so called functional food, which has health promoting activity besides nutritional value. Autochthonous cheeses manufacturing within the Balkan area has very long tradition. These cheeses are produced without using any known starter culture. Therefore, studying all aspects of such cheeses manufacturing is very important. This approach will assure continuation of traditional way of cheeses manufacturing at the original places where they are originally produced. Moreover, research of this kind will open possibility for introduction of autochthonous cheeses production at either semi-industrial or industrial level or their presentation on different markets. The laws on patenting, brand protection or geographical origin declaration are giving the possibilities for protection of autochthonous cheeses. It is not unrealistic to expect that such products will accept on European and other markets. Therefore, the strategy is established to develop the technological process for autochthonous cheese production in the region of mountain Zlatar. The developed technology will be based on the use of starter cultures, which will be constructed of specific lactic acid bacteria (LAB). LAB will be isolated from specific localities on Zlatar, characterized at the molecular genetics level and used for construction of starter cultures

Dominant lactic acid bacteria in artisanal Pirot cheeses of different ripening period

U ovom radu su ispitivana dva sira od svežeg kravljeg mleka različitog perioda zrenja. Sirevi su uzeti iz seoskog domaćinstva u regionu Stare Planine, a proizvedeni su bez dodatka starter kulture. Iz oba sira je izolovano ukupno 106 sojeva bakterija mlečne kiseline. Sojevi su testirani klasičnim fiziološkim i API 50 CH testovima. Takođe je ispitivana proteolitička i antimikrobna aktivnost. Identifikacija bakterija mlečne kiseline je rađena rep-PCR analizom sa (GTG)5 prajmerom. Osam vrsta bakterije mlečne kiseline je izolavano iz sira BGPT9 starog četiri dana (Lactobacillus plantarum, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Enterococcus durans i Leuconostoc garlicum), dok su u siru BGPT10 starom osam meseci bile prisutne samo dve vrste (Lactobacillus plantarum i Enterococcus faecium). Proteolitičku aktivnost je pokazalo 30 izolata iz sira BGPT9, uglavnom enterokoke. Samo jedan izolat iz sira BGPT10 (koji je pripadao vrsti Lactobacillus plantarum) je posedovao delimičnu sposobnost da hidrolizuje β- kazein. Sedam enterokoka iz sira BGPT9 i četiri enterokoke iz sira BGPT10 je proizvodilo antimikrobne substance.In this study two raw cow's milk cheeses of a different ripening period were examined. The cheeses were taken from a country household in the region of mountain Stara Planina and manufactured without adding of starter culture. A total 106 lactic acid bacteria (LAB) strains were isolated from both cheeses. They are tested by classical physiological tests as well as by API 50 CH tests. Proteolytic and antimicrobial activities were done too. Identification of LAB isolates was done by repetitive extragenic palindromic-polimerase chain reaction (rep-PCR) with (GTG)5 primer. The LAB isolates from cheese BGPT9 (four days old) belonged to the eight species of LAB (Lactobacillus plantarum, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Enterococcus durans and Leuconostoc garlicum), while in the BGPT10 cheese (eight months old) only two species were present (Lactobacillus plantarum and Enterococcus faecium). Proteolytic activity showed 30 LAB from BGPT9 cheese, mainly enterococci. From BGPT10 cheese only one isolate (which belonged to the Lactobacillus plantarum species) possessed partial ability to hydrolyze β-casein. Seven enterococci from BGPT9 cheese and four enterococci from BGPT10 cheese produced antimicrobial compounds

Casitone-mediated expression of the prtP and prtM genes in Lactococcus lactis subsp. lactis BGIS29

Casitone added to chemically defined medium (CDM) specifically influenced the regulation of the proteinase activity in the natural isolate Lactococcus lactis subsp. lactis BGIS29. Comparative analysis of the influence of casitone present in CDM on the proteolytic activity of strain BGIS29 and of L. lactis subsp. cremoris strains SK11 and Wg2 indicated that the proteolytic activity of strains BGIS29 and SK11 is casitone-dependent, whereas that of strain Wg2 is not. The regulatory region of the prt genes of strain BGIS29 was cloned and sequenced. The nucleotide sequence of the prt regulatory region of strain BGIS29 was distinctly different from that of L. lactis subsp. cremoris strains SK11 and Wg2. Transcriptional gene fusions with the Escherichia coli β-glucuronidase gene (gusA) were used to study medium-dependent expression of two divergent prtP and prtM promoters of strain BGIS29 (designated PprtP and PprtM, respectively). β-Glucuronidase assays and Northern blot analysis showed that the activities of PprtP and PprtM are controlled by casitone at the transcriptional level.

Analysis of natural isolates of Lactobacilli resistant to bacteriocin nisin

Kolekcija bakterija mlečne kiseline (BMK) je napravljena od mikroorganizama izolovanih iz fermentisanih mlečno-kiselinskih proizvoda dobijenih na tradicionalan način. Iz kolekcije 51 izolat je identifikovan kao Lactobacillus sp. Svi izolovani laktobacili pripadaju grupi mezofilnih sojeva koji dobro rastu na temperaturama od 15°C i 30°C, a ne rastu na temperaturi od 45°C. Testiranje sposobnosti rasta u prisustvu nizina pokazalo je da su izolati BGCGK4, BGHN40, BGBUK2-8, BGBUK2-7 i BGBUK2-16 rezistentni na bakteriocin nizin. U eksperimentu određivanja minimalne inhibitorne koncentracije (MIK) za nizin pokazano je da je najrezistentniji izolat Lactobacillus sp. BGCGK4. Izolat BGBUK2-16, determinisan kao Lactobacillus paracasei subsp. paracasei, produkuje bakteriocin označen kao Bac217 i pokazuje rezistenciju na 8000 IU/ml. Čišćenjem plazmida iz soja BGBUK2-16 dobijena su 2 derivata označena kao BGBUK2-16/K2 i BGBUK2-16/K4. Derivat BGBUK2-16/K2 zadržao je rezistenciju na Bac217 i nizin, ali je izgubio sposobnost sinteze Bac217, dok je derivat BGBUK2-16/K4 pored gubitka sposobnosti sinteze Bac217 postao senzitivan na Bac217 i nizin. Prirodno rezistentni laktobacili se mogu iskoristiti za pripremanje starter kultura u kombinaciji sa nizinom kao konzervansom u cilju kontrolisane mlečno-kiselinske fermentacije.The collection of lactic acid bacteria (LAB) was made by isolation of microorganisms from fermented products traditionally manufactured in different geographical regions (high mountains, river valleys, seaside, etc). Among collected LAB, 51 isolates were identified as Lactobacillus sp. Results showed that all isolated lactobacilli were mesophilic strains, since they grew at 15°C and 30°C but not at 45°C. Testing the ability of isolated lactobacilli to grow in the presence of nisin revealed that Lactobacillus sp. isolates designed BGCGK4, BGHN40, BGBUK2-8, BGBUK2-7 and BGBUK2-16 were resistant to nisin. Determination of the minimal inhibitory concentrations (MIC) for nisin revealed that the most resistant isolate was Lactobacillus sp. BGCGK4. Isolate BGBUK2-16, determined as Lactobacillus paracasei subsp. paracasei, produces bacteriocin, named Bac217 and showed a resistance to 8000 IU/ml of nisin. Plasmid curing of BGBUK2-16 resulted in derivatives BGBUK2-16/K2 and BGBUK2-16/K4. Derivative BGBUK2-16/K2 retained resistance to Bac217 and nisin, but lost the ability to synthesise Bac217. Derivative BGBUK2-16/K4 lost concomitantly the resistance to both Bac217 and nisin

Examination of antimicrobial potential in natural isolates of lactobacillus casei/paracasei group

Cilj ove studije je izučavanje antimikrobnog potencijala 52 prirodna izolata vrste L. casei/paracasei. Učestalost gena koji kodiraju BacSJ (bacSJ2-8/bacSJ2-8i genski klaster), acidocin 8912 (acdT), ABC-transporter (abcT) i pomoćni protein (acc) su takođe izučavani. Genski klaster bacSJ2-8/bacSJ2-8i prisutan je kod 49 (94.23%), a acdT kod 41 (78.85%) od 52 testirana soja. Četrdeset sojeva (76.92%) poseduje oba analizirana gena. Interesantno je da samo 17 sojeva (32.69%) koji poseduju bacSJ2-8/bacSJ2-8i genski klaster i/ili acdT gen proizvode bakteriocine. Soj L. paracasei BGNK1-62 poseduje bacSJ2-8/bacSJ2-8i genski klaster, ali ne proizvodi bakteriocin BacSJ što je verovatno posledica nedostatka abcT i acc gena. Nakon transformacije soja BGNK1-62 konstruktom pA2A koji poseduje abcT i acc gene ostvarena je proizvodnja bakteriocina BacSJ. Osim toga, utvrđeno je da soj L. paracasei BGGR2-66 proizvodi nov bakteriocin označen kao BacGR, koji je biohemijski okarakterisan, a određena je i njegova N-terminalna sekvenca.The aim of this study was to investigate the antimicrobial potential of 52 natural isolates of Lactobacillus casei/paracasei. The incidence of relevant genes encoding BacSJ (bacSJ2-8/bacSJ2-8i gene cluster), acidocin 8912 (acdT), ABC-transporter (abcT) and accessory protein (acc) was also studied. These genes were found to be widespread amongst the analyzed L. casei/paracasei strains. The bacSJ2-8/bacSJ2-8i gene cluster was present in 49 (94.23%) and acdT in 41 (78.85%) of the 52 tested strains. Forty of these strains (76.92%) harbored both analyzed genes. Interestingly, only 17 strains (32.69%) with the bacSJ2-8/bacSJ2-8i gene cluster and/or the acdT gene showed bacteriocin production. Strain L. paracasei BGNK1-62 contained the bacSJ2-8/bacSJ2-8i gene cluster, but did not produce bacteriocin BacSJ possibly due to absence of the abcT and acc genes. Hence, these genes were introduced into BGNK1-62 by transformation with constructed plasmid pA2A, after which BacSJ was produced. In addition, it was found that L. paracasei BGGR2-66 produced new bacteriocin designated as BacGR that was biochemically characterized and its N- terminal sequence was determined

Characterisation of the exopolysaccharide (EPS)-producing Lactobacillus paraplantarum BGCG11 and its non-EPS producing derivative strains as potential probiotics

Traditional fermented foods are the best source for the isolation of strains with specific traits to act as functional starters and to keep the biodiversity of the culture collections. Besides, these strains could be used in the formulation of foods claimed to promote health benefits, i.e. those containing probiotic microorganisms. For the rational selection of strains acting as probiotics, several in vitro tests have been proposed. In the current study, we have characterized the probiotic potential of the strain Lactobacillus paraplantarum BGCG11, isolated from a Serbian soft, white, homemade cheese, which is able to produce a >ropy> exopolysaccharide (EPS). Three novobiocin derivative strains, which have lost the ropy phenotype, were characterized as well in order to determine the putative role of the EPS in the probiotic potential. Under chemically gastrointestinal conditions, all strains were able to survive around 1-2% (10 6-10 7cfu/ml cultivable bacteria) only when they were included in a food matrix (1% skimmed milk). The strains were more resistant to acid conditions than to bile salts and gastric or pancreatic enzymes, which could be due to a pre-adaptation of the parental strain to acidic conditions in the cheese habitat. The ropy EPS did not improve the survival of the producing strain. On the contrary, the presence of an EPS layer surrounding the strain BGCG11 hindered its adhesion to the three epithelial intestinal cell lines tested, since the adhesion of the three non-ropy derivatives was higher than the parental one and also than that of the reference strain Lactobacillus rhamnosus GG. Aiming to propose a potential target application of these strains as probiotics, the cytokine production of peripheral blood mononuclear cells (PBMC) was analyzed. The EPS-producing L. paraplantarum BGCG11 strain showed an anti-inflammatory or immunosuppressor profile whereas the non-ropy derivative strains induced higher pro-inflammatory response. In addition, when PBMC were stimulated with increasing concentrations of the purified ropy EPS (1, 10 and 100μg/ml) the cytokine profile was similar to that obtained with the EPS-producing lactobacilli, therefore pointing to a putative role of this biopolymer in its immune response. © 2012 Elsevier B.V.This work was financed by the Spanish Ministry of Science and Innovation (MICINN) through the project AGL2009-09445 (FEDER European Union funds) and by the Ministry of Education and Science of the Republic of Serbia grant No. 173019.Peer Reviewe