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Correlative phenotypic expressions of plasmids for proteolytic activity and bacteriocin
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production in Lactococcus lactis isolated from raw and skimmed milk
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Ajunwa O.M.1,3, Odeniyi O.A.3, Adeleke A.J.1,3*, Nwanekwu K.E.2 and Obiukwu C.E2
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1. Department of Microbiology, Modibbo Adama University of Technology, Yola,
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Adamawa State, Nigeria
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2. Department of Microbiology, Imo state university, Owerri, Imo state, Nigeria
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3. Department of Microbiology, University of Ibadan, Oyo State, Nigeria
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Corresponding author*:
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Name: Adeleke A.J.
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Postal Address: Department of Microbiology, Modibbo Adama University of Technology,
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Yola, Adamawa State, Nigeria.
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Phone Number: +2348060260571
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Email Address: lekejohnson2222@mautech.edu.ng
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Keywords: Plasmids, bacteriocin, proteinase, Lactococcus lactis, chromosome
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ABSTRACT
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Lactococcus lactis is a major member of the Lactic Acid Bacteria (LAB) group and it is
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known to produce bacteriocins and proteinases. A total of thirty two (32) L. lactis strains
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were isolated from spontaneously fermented raw cattle milk and skimmed milk. Only four
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isolates had correlative proteinase and bacteriocin production abilities with evident zones of
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inhibition and zones of proteolysis (above 5mm diameter). L. lactis RCM9 had 3 small sized
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plasmids (1kbp, 3.5kp and 4.5kbp). L. lactis RCM 21 also possessed 3 plasmids (6kbp, 9kbp
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and >10kbp), while L. lactis SKM 14 possessed plasmid >10kbp and L. lactis RCM 15
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possessed no plasmid. A combination of plasmid curing treatments of novobiocin (10µg/ml),
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ethidium bromide (40µg/ml) and incubation at 40oC resulted in an entire loss of plasmids
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after re-conducting the profiling. Phenotypic assertions on the plasmid related
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bacteriocinogenicity and proteinase activity of both plasmid cured and uncured isolates
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revealed that bacteriocin and proteinase production in L. lactis RCM 9 and L. lactis RCM 21
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were not plasmid mediated but chromosomal. However, plasmids were responsible for
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similar traits in L. lactis SKM14 and L. lactis RCM 21. This showed the inconsistency in
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plasmid presence and activity within strains.
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INTRODUCTION
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The fact that lactic acid bacteria (LAB) are one of the most industrially important organisms
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known to man cannot be overemphasised. They have been identified as a major set of food
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grade organisms and as a result are extensively applied as starter culture in the production of
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a large variety of fermented products. In recent times, their direct application has been
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heightened by the fact that they are generally regarded as safe for human consumption. In this
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regard, great deals of scientific investigations have been initiated into the methods of
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industrialisation and commercialisation of the applications of the beneficial properties of
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LAB. These methods have spanned fields of molecular biology, food microbiology,
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biotechnology, bioengineering, and applied microbiology, and have been subject to
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improvement with respect to scale-up, optimisation and maximisation of the industrial
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potentials of LAB.
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A major importance of LAB that has ascribed to it this level of industrial importance is the
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production of enzymes and antimicrobial metabolites. Of all the antimicrobial substances
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produced by LAB, bacteriocins have been accorded the most attention. This is largely due to
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their specie specific inhibition and unique molecular mechanisms of action. Quantifiable
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evidence by earlier investigators has proven the efficacy of bacteriocins from LAB against an
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array of both Gram positive and Gram negative organisms (Suskovic et al., 2010). Enzyme
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production in LAB is also of major reckon as enzymes like proteinases have been derived
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from LAB metabolism (Kok, 1987, Kojic et al., 1991, Hill and Gasson, 1986). Several genera
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make up the LAB group, and each has unique peculiarities. One of the primary genuses of
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LAB is the Lactococcus spp. This genus was formerly grouped with the streptococci. These
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organisms are employed as dairy starters in the manufacture of fermented milk. Lactococcus
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spp also produce bacteriocins (antimicrobial peptides) like nisin, lactococcin, and diplococcin
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which are of great industrial importance (Geiss et al., 1983). Studies on the genetic
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mechanisms of bacteriocin production in LAB, have illuminated the role of plasmids. With
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respect to the industrial value of the bacteriocins produced by lactococci, L. Lactis has shown
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to be the most bacteriocinogenic of all Lactococcus spp (Kojic et al., 2005).
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According to Gasson (1983) and Gireesch et al. (1992), plasmids are commonly found in
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lactococci. The varying numbers of plasmids range in size from 3 to 130kb, and have been
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determined in different strains of lactococci. In this same regards, some lactococci have also
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been found not to contain any plasmids (Nissen-Meyer et al., 1992). To gain a favourable
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insight into the lactococcal plasmids biology, two mechanisms have been identified to be
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responsible for their replication – theta replication (Kiewiet et al., 1993) and rolling circle
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replication (Leehouts et al., 1991). Khan (1997) presumptively stated that the rolling circle
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mechanism was restricted to relatively small plasmids whose functions still remained unclear.
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As earlier stated, a number of lactococcal strains produce bacteriocins. Geiss et al. (1983)
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conducted an extensive survey and showed that 5% of a total of 280 tested lactococcal strain
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produced bacteriocins. Among the bacteriocins identified is nisin, the most prominent of all
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peptide antimicrobials. Further studies on the plasmid-mediated nisin production revealed the
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activities of two chromosomally located conjugative transposons (Tn 5301 and Tn 5276)
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identified to be present in L. lactis as responsible for the production of the peptide (Rauch
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and de Vos 1992; Dodd et al., 1990; Horn et al., 1991). In view of the conjugative property
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of plasmids, the ability for Lactococcus lactis to produce other bacteriocins is possible as the
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plasmids for bacteriocins like diplococcin or lactococcins have proven to be transferable via
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conjugation (Harmon and McKay, 1987). Other studies on bacteriocin production by
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different strains of L. lactis have revealed the presence of lactococcin A gene cluster from the
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plasmid pNP2 (Stoddard et al., 1992) and the presence of 3 different bacteriocins, namely
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lactococcin A, lactococcin B and lactococcin M/N in L. lactis subsp cremoris 9B4 (Van
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Belkum et al., 1991)
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The presence of lactococci in milk can be attributed to their ability to metabolise the proteins
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and other compounds inherent in the environment. To ably convert the protein, a compact
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proteolytic system has been known to be responsible. Kojic et al. (2005) explained that cell-
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wall-bound extracellular proteinases are implicated in the crucial role of initially hydrolysing
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the proteins. Genetic studies by Kok (1990) and Nissen-Meyer et al. (1992) identified that the
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gene coding for extracellular proteinase production in lactococci was located on a plasmid.
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This work thus seeks to investigate the abilities of plasmids in L. lactis to code for protein
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hydrolysing enzymes and also for bacteriocin production. The study reports on the genetic
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relationship between the phenotypic traits of bacteriocin proteinase production as obtained in
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the plasmids. The consideration of the inductive effects of proteins and protein based
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environments on protein hydrolysis was considered as raw cattle milk and skimmed milk
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were used as the primary isolation environment of L. lactis used in the study.
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MATERIALS AND METHODS
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Isolation and characterisation of L. lactis
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Freshly drawn raw cattle milk and commercially available skimmed milk were obtained in
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containers and allowed to undergo spontaneous fermentation for 72 hours. Serial dilutions
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were made and 1ml of 10-5-10-10 as aliquots were aseptically plated out on MRS agar and
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M17 agar media via pour and spread plate technique. Aerobic and anaerobic methods of
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incubation were observed and suspected LAB colonies were subcultured for pure colonies.
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To characterise the isolates, macroscopic observations included parameters such as colony
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shape, size, elevation, type of growth, pigmentation, spore staining, catalase test, nitrate
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reduction, growth in different Sodium Chloride concentrations, growth at different
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temperatures, carbohydrate fermentation tests, motility, homolactic and heterolactic
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properties. L. lactis isolate were identified after series of biochemical tests and screening and
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subsequently subjected to antagonistic studies.
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Preliminary antagonistic studies
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To ascertain the preliminary antagonistic state of L. lactis isolates, the isolates were prepared
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in broth and aliquots were made into sterile bottle. Bacteriocin sensitive Lactobacillus casei
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isolates were obtained from the Federal Institute of Industrial Research (FIIR) and used as the
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test organism. The agar well diffusion method of Tagg and McGiven (1971) with
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modifications by Ajunwa (2011) was carried out. The L. lactis in broth was transferred at
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50µl quantities into the wells. After incubation for 48 hours, the plates were observed for
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zones of inhibition. Plates with at least 5mm diameters were used and their labelled stocks
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were selected for assay for bacteriocin production.
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Assay for bacteriocin production
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To detect bacteriocin production, broth culture of antagonistic L. lactis were freshly prepared
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and incubated overnight at 30oC. The liquid broth was centrifuged at 10000 r.p.m for 5
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minutes. M17 soft agar (0.7% w/v) was used to propagate the bacteriocin sensitive L. casei
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wells were made in the lawn of soft agar, and 50µl aliquots of filtered supernatant of
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overnight culture were poured into the wells. The appearance of a clear zone representing
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inhibition of growth of the sensitive L. casei around a well implied a positive signal for
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bacteriocin production. Filtered supernatant of overnight culture of bacteriocin non-producer
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cells of L. lactis were used as negative control.
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Assay for proteinase activity
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To test proteinase activity, all isolates of L. lactis were induced by growing on milk-citrate
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agar (MCA) plates containing 4.4% reconstituted skimmed milk, 0.8% Na-citrate, 0.1% yeast
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extract, 0.5% glucose and 1.5% agar (w/v). The cells were grown in broth of the same
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medium, and the detection of caseinolytic/proteinolytic activity was performed using a
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casein-based medium for conduction of agar well diffusion assay. Any form of clearing
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around the colonies implied a positive signal for proteinase production. They were measured
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and representative cultures were properly labelled.
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Plasmid isolation and plasmid curing
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Plasmid isolation was carried out according to the method of Anderson and Mckay (1983)
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with adaptations to the method of Jamuna et al. (2010). The presence of plasmids was
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checked via agarose gel electrophoresis with 0.7% agarose in the presence of Tris-borate
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buffer at 100volts for 4hrs. 0.5µg/ml of Ethidium Bromide was used for staining the gel
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which as subsequently viewed under UV light on a transluminator and photographed.
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Plasmid size was determined according to the method of Jamuna et al. (2010) using the
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logarithmic conversion of the molecular weight of standard DNA molecular size markers run
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concurrently with their respective mobility.
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Plasmid curing was conducted with modifications according to the method of Kojic et al.
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(2005). It was achieved by growing the cells in prewarmed M17 broth (40oC) containing
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ethidium bromide (40µg/ml) and novobiocin (10µg/ml). The cells were collected by
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centrifugation every 2 hours after incubation at 40oC. The collected cells were resuspended in
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the same volume of M17 broth containing novobiocin and ethidium bromide, and the process
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was repeated severally. Subsequently, 0.1ml aliquots of cell broth were plated on M17 agar
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plates and incubated at 30oC for 48hours. After incubation, plates were overlaid with M17
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soft agar containing indicator L. casei and incubated overnight at 30oC. Colonies were
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checked for inhibition against the indicator organism. Earlier identified bacteriocin producing
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cells without zones of inhibition after curing were termed as ‘cured’. Bacteriocin production
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was carried out with the cured and uncured cells
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Bacteriocin production
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For inducement of bacteriocin production a chemically defined and modified MRS broth
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containing reduced concentration of peptone (0.5% w/v) and glucose (0.25% w/v) was used
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to propagate the LAB strains at 30oC for 72hours. An anaerobic environment was also
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employed during incubation. Cells were removed from the stationary phase of the growth
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broth medium by centrifugation at 10000 rpm for 10 minutes at 4oC. The supernatant fluid
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was adjusted to pH 6.5 and then treated with 5mg/ml catalase. The supernatant was then filter
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sterilized through a 0.4µm pore size membrane filter and the product was designated as
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‘crude bacteriocin’. Filtered supernatant of cultures of bacteriocin-non-producer cells was
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used as negative control. Samples of the extracted bacteriocin fluid were subjected to the
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action of Proteinase K (0.5 mg/ml) to ascertain the protein nature of the fluids.
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Propagation of plasmid cured isolates
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The plasmids cured isolates of L. lactis were repropagated on M17 agar and MRS agar and
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subjected to assay for bacteriocin production. They were also subjected to proteinolytic
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enzyme assay. The resulting zones of inhibition were subsequently measured.
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RESULTS
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Macroscopically, the colonies were cream coloured on MRS medium with smooth surfaces.
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The edges were entire, and the isolates were translucent. The colonies were small and round.
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Microscopic analysis showed the isolates to be cocci with positive Gram reaction. They were
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negative to nitrate reduction tests, spore test, catalase tests, and grew in 5% NaCl. They also
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grew at 10oC but no growth was observed at 45oC. Biochemically, they utilised glucose,
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maltose, sucrose, mannitol, galactose, lactose, and xylose, but were unable to utilise
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arabinose. They were homofermentative in nature producing lactic acid as the primary
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metabolic product.
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Thirty two strains of L. lactis were isolated from both raw cattle milk and commercially
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available skimmed milk. Isolates from raw cattle milk were designated with RCM while
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SKM was used for isolates from skimmed milk. All the isolates were subjected to primary
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antagonistic studies against L. casei, and proteinase assay. Twenty of the isolates were
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positive for proteinase production. Out of the total number only four isolates showed zones of
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inhibition (above 5mm) against the test organism which was indicative for bacteriocin
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production. The four isolates were also positive for proteinase production, indicating
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phenotypic correlation in proteinase and bacteriocin expressions. Figure 1 shows the zones of
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inhibition (mm) of the L. lactis isolated from the two milk sources against non-
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bacteriocinogenic L. casei as test organisms, and zones of clearing (mm) of proteolysis.
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Plates showing phenotypic expressions of bacteriocin and proteinase activity were illustrated
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by Figures 2 and 3.
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Plasmid profiling carried out on the four bacteriocinogenic and proteinase producing L. lactis
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isolates revealed that L. lactis RCM 9 possessed 3 plasmids of approximately 1kbp, 3.5kbp,
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4.5kp. L. lactis SKM 14 possessed plasmid greater than 10kbp. Three plasmids were present
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in L. lactis RCM 21 with weights of 6kbp, 9kbp, and >10kbp, while L. lactis RCM 15
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possessed no plasmid. Plasmid curing conducted on all four isolates showed a complete
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elimination of all plasmids which was evident by loss of plasmids after a plasmid profile was
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re-conducted. Figure 4 shows the plasmid profile of all four L. lactis isolates.
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Plasmid cured isolates were subjected to phenotypic determinations of bacteriocin activity
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and proteolysis. It was observed that plasmid cured L. lactis RCM 9 and L. lactis RCM 15
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still showed bacteriocin and proteolytic activity, proving that the bacteriocin and proteolytic
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activity were not plasmid mediated. However, L. lactis SKM 14 and L. lactis RCM 21
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showed negative bacteriocin and proteolytic activity after plasmid curing proving the fact that
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the traits were plasmid associated and probably plasmid mediated.
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DISCUSSION
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The isolation environments (raw cattle milk and skimmed milk) generally aided the luxuriant
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growth of L. lactis with no specific differences in the expressional abilities of bacteriocin and
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proteinase activities of the isolates derived from the two environments. The results from this
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work showed that there was a correlation in the expression of proteinase activity with
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bacteriocin mediated inhibition in some L. lactis strains. The effects of plasmids in relation to
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phenotypic characters has thrown open a major form of inquiry in molecular biology,
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especially with respect to bacteria of industrial importance. This proved to be in direct
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consonance with reports by Sieuwerts et al. (2008), and Suskovic et al. (2010). However, the
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interplay in plasmid mediation of these characteristics can be based on factors like; the size of
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plasmids and the number of plasmids present within a cell as evidenced by analysis by Turner
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(2004).
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Plasmids occurring in the bacteriocin and proteinase producing cells of the L. lactis isolates
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ranged from 1kbp to >10kbp. L. lactis RCM 9 which possessed the small sized plasmids
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(1kbp, 3.5kbp, and 4.5kbp) proved to possess bacteriocin and proteinase production traits
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independent of the plasmid possessed. This case proves that the plasmids coded for traits
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other than bacteriocin or proteinase production, and that the bacteriocin and proteinase
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production genes were chromosomally located. With regards to the small size of the plasmids
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observed in the isolate, Khan (1997) explained that the existence of relatively small
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lactococcal plasmids and their functions have been relatively unclear. L. lactis RCM 15
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possessed no plasmids and thus its bacteriocinogenicity and proteinase production traits were
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not affected by plasmid curing. This confirms reports about the unsteady genetic
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configuration of traits like bacteriocin production in Lactococcus species (Nettles and
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Barefoot, 1993).
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L. lactis SKM 14 and L. lactis RCM 21 possessed bacteriocin and proteinase production traits
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which were plasmid mediated. They harboured plasmids of varying molecular weights, but
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common to both were plasmids >10kbp. This is in line with findings by Kojic et al. (2005)
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which showed L. lactis isolates possessing very large plasmids ( > 100 kilo bases) coding for
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proteinase and lactococcin production. The sizes of the plasmids recorded in this work are
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relatively small compared to those obtained by Kojic et al. (2005), however, further work on
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the specific characterisation and classical determination of the proteinolytic enzymes and
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bacteriocins produced (based on molecular weight determination and purification methods)
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will aid in conclusive exposition of the phenotypic traits of plasmid bearing L. lactis strains
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with respect to the plasmid size.
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In general, plasmids are highly unstable and the clarity in their reported functions has been
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plagued by a plethora of laboratory and natural factors. Nevertheless, conscious attempts to
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preserve the plasmid-mediated beneficial traits of industrially important bacteria are
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consistently in motion, thus requiring extended research.
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