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110209 composition medium_culture_media (4) Document Transcript

  • 1. Journal of Biotechnology 121 (2006) 54–61 Optimization of culture conditions and medium composition for the production of micrococcin GO5 by Micrococcus sp. GO5 Mi-Hee Kim 1 , Yoon-Jung Kong, Hong Baek, Hyung-Hwan Hyun ∗ Department of Biological Science and Biotechnology, Hankuk University of Foreign Studies, Yongin 120-749, Republic of Korea Received 8 February 2005; received in revised form 17 June 2005; accepted 27 June 2005Abstract To enhance the production of micrococcin GO5, a bacteriocin produced by Micrococcus sp. GO5, cultivation conditions andmedium composition were optimized. The optimal initial pH and temperature for bacteriocin production were 7.0–9.0 and 37 ◦ C,respectively. Micrococcus sp. GO5 displayed the highest micrococcin GO5 activity when grown in modified MRS medium thatcontained lactose or sucrose, rather than glucose, as a carbon source. The maximum bacteriocin activity was obtained in modifiedMRS medium containing 0.5% tryptone and 1.0% yeast extract as nitrogen sources instead of the other nitrogen sources presentin MRS medium. Bacteriocin production was greatly affected by the concentration of K2 HPO4 ; strain GO5 produced eight-foldmore bacteriocin in medium containing 2.0–2.5% K2 HPO4 than in medium containing 0.2% K2 HPO4 . The optimal concentrationof MgSO4 ·7H2 O for bacteriocin production was 0.5%. The production of micrococcin GO5 was increased 32-fold in shake flaskculture and 16-fold in a bioreactor using the optimized medium (TY medium), compared with culturing in MRS medium.© 2005 Elsevier B.V. All rights reserved.Keywords: Bacteriocin; Micrococcin; Micrococcus sp.; Kimchi; Production1. Introduction microorganisms, and they are therefore potentially use- ful as natural replacements for synthetic food preserva- Bacteriocins, which are produced by certain strains tives. Nisin, a bacteriocin produced by certain strainsof lactic acid bacteria and some other prokaryotes, are of Lactococcus lactis subsp. lactis, is one of the mostproteinaceous antimicrobial agents (Tagg et al., 1976). intensively studied lantibiotics, due to its industrialSome bacteriocins inhibit food spoilage and pathogenic application and potential for other uses. It is composed of 34 amino acids and has a broad spectrum of activ- ity and extreme heat stability at low pH (Choi et al., ∗ Corresponding author. Tel.: +82 31 330 4279; 2000; Delves-Broughton, 1990). Nisin is generally rec-fax: +82 31 330 4566. E-mail address: hhhyun@san.hufs.ac.kr (H.-H. Hyun). ognized as safe (GRAS); it is commercially produced 1 Present address: R&D Center, Daewon Pharm. Co. Ltd., Seoul by microbial cultivation and has been widely used as a143-150, Republic of Korea. food preservative in many countries.0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2005.06.022
  • 2. M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 54–61 55 The production of bacteriocins is, in general, closely used as the indicator strain for the bacteriocin activ-associated with bacterial growth because bacteriocins ity assay. Stock cultures were maintained at −70 ◦ C inare synthesized only during the growth of the produc- MRS medium (BD, Sparks, MD) containing 20% (v/v)ing organism; bacteriocin activities decrease more or glycerol. Before use in experiments, the stock culturesless sharply at the end of the growth phase as a result were activated by cultivation twice in MRS medium atof degradation by proteases (De Vuyst and Vandamme, 37 ◦ C for 6 h.1994; Green et al., 1997; Hur et al., 2000). Bacteriocinproduction is also affected by the medium composition 2.2. Media and culture conditionsand culture conditions, such as pH, temperature andagitation. Therefore, the optimization of environmen- Micrococcus sp. GO5 was cultured in MRStal conditions is very important for the enhancement of medium. The indicator strain was cultured in TGYbacteriocin production. The effect of medium compo- medium (Kim et al., 2005). The seed cultures of Micro-sition on the production of bacteriocins, such as nisin coccus sp. GO5 were prepared by cultivating the cells(De Vuyst and Vandamme, 1992, 1993; Matsusaki et in 500 ml Erlenmeyer flasks that contained 50 ml ofal., 1996), pediocin (Biswas et al., 1991), enterocin MRS medium and were shaken at 250 rpm and main-(Parente and Hill, 1992), lactococcin (Parente et al., tained at 37 ◦ C until the late exponential growth phase.1993) and mesenterocin (Daba et al., 1993) has been For flask culture, 0.5 ml of the seed culture was inocu-reported. Nisin production is affected by carbon source lated into 50 ml of the appropriate medium in a 500 mlregulation, nitrogen sources and phosphorus (De Vuyst Erlenmeyer flask. The initial pH of the flask cultureand Vandamme, 1992, 1993). The pH of the medium was adjusted to 6.8 with 5N NaOH, and the flasks wereplays an important role in the production of bavaricin incubated at 37 ◦ C with shaking at 250 rpm.(Kaiser and Montville, 1993), nisin (De Vuyst and Van- To test the effect of the initial pH on bacteriocindamme, 1992) and lactococcin (Parente et al., 1993, production, MRS media were adjusted to different pH1994). values (pH 4.0–9.0) with 5N NaOH or 5N HCl. To test In our previous paper (Kim et al., 2005), we reported the influence of carbon sources, each carbon sourcethat Micrococcus sp. GO5, isolated from traditional was added as 2% of the MRS medium, replacing theKorean kimchi prepared by cultivation of a mixture 2% glucose. To study the effect of different nitro-containing green onion, spices and other ingredients, gen sources, a modified MRS medium was used asproduced a bacteriocin, which was tentatively named a basal medium, yeast extract, beef extract, peptonemicrococcin GO5. Micrococcin GO5 is very similar and ammonium citrate, were omitted and 2% glucoseto nisin in its spectrum of inhibitory activity, heat sta- was replaced with 2% lactose. The basal medium wasbility and pH stability, but its structure is unlike that supplemented with each nitrogen source (1%) or a mix-of nisin; it has a different molecular weight (5.0 kDa), ture of multiple nitrogen sources. To investigate thedifferent amino acid composition, and different N- influence of the phosphate concentration, different con-terminal amino acid sequence. This paper describes centrations of K2 HPO4 were added to the above basalhow the optimization of growth parameters, such as medium, which was supplemented with 0.5% tryp-temperature, medium composition and pH, resulted in tone and 1.0% yeast extract. The experiments to deter-the enhancement of micrococcin GO5 production by mine the influence of magnesium sulfate concentrationMicrococcus sp. GO5. were carried out using a modified basal medium that contained lactose (20 g l−1 ), tryptone (5 g l−1 ), yeast extract (10 g l−1 ), K2 HPO4 (22.5 g l−1 ), sodium acetate2. Materials and methods (5 g l−1 ), Tween 80 (1 g l−1 ) and different concentra- tions of MgSO4 ·7H2 O. All experiments to evaluate2.1. Bacterial strains culture conditions and medium composition except for the influence of temperature were performed by shake The micrococcin GO5-producing strain used in this flask culture. The experiments on temperature effectstudy was Micrococcus sp. GO5, previously isolated and the cultivation time course studies were conductedfrom kimchi. Micrococcus flavus ATCC 10240 was using a 5 l bioreactor (Kobiotech Co., Inchon, Korea),
  • 3. 56 M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 54–61of which pH and temperature were controlled, and that samples to be spotted were serially diluted two-fold,contained 3 l of culture medium and was agitated at and the reciprocal of the highest inhibitory dilution was700 rpm and aerated at 1 vvm. The temperature effect used to calculate the arbitrary activity units (AU) peron bacteriocin production was determined in a biore- millilitres. The un-inoculated media were also testedactor containing MRS medium, which was maintained for inhibitory zones as a control. All experiments wereat different temperatures (25, 30 and 37 ◦ C) and con- performed in duplicate, and the results are the meanstrolled at pH 7.0. The cultivation time course studies of duplicate trials.to compare MRS medium and TY medium were con-ducted in a 5 l bioreactor that was maintained at 37 ◦ C 2.4. Analytical methodsand controlled at pH 6.0. TY medium contained lactose(20 g l−1 ), tryptone (5 g l−1 ), yeast extract (10 g l−1 ), For the determination of culture turbidity, cul-K2 HPO4 (22.5 g l−1 ), sodium acetate (5 g l−1 ), Tween ture broths were appropriately diluted with distilled80 (1 g l−1 ) and MgSO4 ·7H2 O (5 g l−1 ). water, and the optical densities were measured at 660 nm using a spectrophotometer (Shimadzu UV-2.3. Micrococcin GO5 assay 1601, Japan). The un-inoculated media were used as a blank. The antimicrobial activity of micrococcin GO5 inculture medium was measured according to the follow-ing procedures. Samples were adjusted to pH 6.0 with 3. Results and discussion5N NaOH, centrifuged at 8000 × g for 10 min at 4 ◦ C,and then filter-sterilized through a 0.45 ␮m pore size, 3.1. The initial pH, cell growth and micrococcinmixed cellulose ester membrane (Advantec Mfs. Inc., GO5 productionJapan). Micrococcin GO5 activity was assayed accord-ing to the spot-on-lawn method (Tagg et al., 1976). Table 1 shows the dependence of cell growth andCells of Micrococcus flavus ATCC 10240 were grown bacteriocin production in Micrococcus sp. GO5 on thein TGY medium at 37 ◦ C for 12 h, and then mixed with initial pH of the medium. The species displayed poorTGY soft agar (0.7%). Three milliliters of the mix- growth at initial pH values lower than 5.0, and the finalture containing 107 cells was overlaid onto 1.5% TGY cell concentration reached a maximum when the initialagar plates. Ten microliters of the filter-sterilized bac- pH was 9.0. This implies that the higher the initial pHteriocin sample was spotted on a TGY agar plate, and value in a shake flask culture without pH control, thethe plate was incubated at 37 ◦ C for about 12 h until greater the cell growth will be before the pH drops bythe inhibition zone was clearly visible. The bacteriocin acid production to below pH 5.0 and growth is curtailed.Table 1Influence of initial pH on cell growth and micrococcin GO5 production in Micrococcus sp. GO5aInitial pH of medium Fermentation time (h) Final pH Growth (OD660 ) Micrococcin GO5 activity (AU ml−1 )b4.0 8 4.14 0.7 85.0 8 4.91 1.8 86.0 8 4.90 6.2 646.5 8 4.86 9.5 647.0 7 4.93 11.0 1288.0 7 4.93 12.0 1289.0 7 5.21 14.0 128Data are means of duplicates. Standard errors were less than 5% of the means. a Cells were grown at 37 ◦ C in a 500 ml Erlenmeyer flask containing 50 ml of MRS medium, which was shaken at 250 rpm. Cell growth, pHand bacteriocin activity were measured at time intervals, and the values when bacteriocin activity was maximum are presented. b AU ml−1 represents the reciprocal of the highest inhibitory dilution in the two-fold dilution assay of bacteriocin.
  • 4. M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 54–61 57Fig. 1. Effect of temperature on cell growth (open symbols) and Fig. 2. Effect of carbon sources on cell growth (open bars) and micro-micrococcin GO5 production (closed symbols) by Micrococcus sp. coccin GO5 production (closed bars) in Micrococcus sp. GO5. CellsGO5. Cells were grown in a bioreactor that contained 3 l of MRS were grown at 37 ◦ C in a 500 ml Erlenmeyer flask containing 50 ml ofmedium, was controlled at pH 7.0, and was maintained at 25 ◦ C MRS medium supplemented with 2% of each carbon source instead( , ), 30 ◦ C ( , ) and 37 ◦ C (᭹, ). Data points are means of of glucose. Cell concentration and bacteriocin activity were mea-duplicates. Standard errors were less than 5% of the means. sured at time intervals, and the maximum values are presented. Data bars are means of duplicates. Standard errors were less than 5% ofThe production of micrococcin GO5 was maximized the means.when the initial pH was between 7.0 and 9.0. In gen-eral, pH is known to be important to cell growth as were reported for organisms producing nisin and nisin-well as to bacteriocin production because aggregation, like bacteriocins (Cheigh et al., 2002; Matsusaki et al.,adsorption of bacteriocin to the cells, and/or prote- 1996).olytic degradation depend on pH and can affect thebacteriocin activity in culture supernatants (Cheigh et 3.3. Carbon sources and micrococcin GO5al., 2002; De Vuyst et al., 1996; Parente et al., 1994; productionVerellen et al., 1998). To determine the optimal pHs forgrowth and bacteriocin production in Micrococcus sp. The effect of the carbon source on cell growth andGO5, further studies in a pH-controlled culture vessel bacteriocin production was determined using MRSwill be necessary. medium supplemented with 2% of different carbon sources in place of glucose. As shown in Fig. 2, the3.2. Temperature, cell growth and micrococcin highest growth was observed in media containing mal-GO5 production tose; glucose, fructose, lactose and sucrose were also suitable carbon sources for growth. However, the high- The effect of temperature on cell growth and micro- est bacteriocin activity was obtained in MRS mediumcoccin GO5 production was tested using a bioreactor with lactose or sucrose (256 AU ml−1 ); the activitycontaining 3 l of MRS medium that was controlled at was twice that obtained with glucose, maltose or fruc-pH 7.0 and maintained at different temperatures (25, tose. Glucose and sucrose were reported to be suitable30 and 37 ◦ C). The results in Fig. 1 show that both carbon sources for nisin Z production (Matsusaki etthe growth rate and the production rate of the bacte- al., 1996), lactose for nisin-like bacteriocin productionriocin increased with an increase of temperature. The (Cheigh et al., 2002) and glucose for Streptococcin A-highest activity of micrococcin GO5 (128 AU ml−1 ) FF22 production (John and Ingrid, 1991).was obtained at 6 h after cultivation at 37 ◦ C, whichwas also optimal for growth. At 40 ◦ C, cell growth and 3.4. Nitrogen sources and micrococcin GO5bacteriocin activity were negligible (data not shown). productionThe optimal temperature for growth and bacteriocinproduction were the same in the case of Micrococ- In order to assess the effect of the nitrogen source oncus sp. GO5, but differences in optimal temperatures growth and micrococcin GO5 production, yeast extract,
  • 5. 58 M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 54–61beef extract and peptone were eliminated from theMRS medium, and glucose was replaced by 2% lac-tose. This basal medium was supplemented with eachdifferent nitrogen source (1%). Cell growth was greatlyenhanced by the addition of beef extract, peptone, tryp-tone, soytone or yeast extract to the basal medium(Fig. 3). The greatest cell growth was observed in cellsgrown in medium with yeast extract (OD660 = 8.4); thecell concentration was almost two-fold that observedin basal medium with beef extract. This implies thatyeast extract contains nitrogen sources and growth fac-tors sufficient to support the growth of Micrococcussp. GO5. However, the highest activity of micrococ-cin GO5 was observed when the cells were grown in Fig. 3. Effect of nitrogen sources on cell growth (open bars) and micrococcin GO5 production (closed bars) in Micrococcus sp. GO5.the tryptone-containing medium, and the bacteriocin Cells were grown at 37 ◦ C in a 500 ml Erlenmeyer flask contain-activity was at least twice those observed with the ing 50 ml of MRS medium supplemented with 1% of each nitrogenmedia containing other nitrogen sources. Because yeast source instead of peptone, yeast extract and beef extract. Cell con-extract was the optimal nitrogen source for growth and centration and bacteriocin activity were measured at time intervals,tryptone was most suitable for micrococcin GO5 pro- and the maximum values are presented. Data bars are means of dupli- cates. Standard errors were less than 5% of the means.duction, the use of multiple nitrogen sources mightenhance both growth and bacteriocin production. How-ever, as shown in Table 2, cell growth and bacteriocin peptone and 0.5% beef extract, but the activity of micro-production were only slightly promoted by the com- coccin GO5 in this medium was equal to that obtainedbination of multiple nitrogen sources. The highest cell with the basal medium supplemented with 0.5% tryp-concentration was obtained when the basal medium tone and 1.0% yeast extract or with 0.5% tryptone andwas supplemented with 0.5% tryptone, 0.5% proteose 1.0% beef extract. Therefore, 0.5% tryptone and 1.0%Table 2Effect of the combination of multiple nitrogen sources on growth and micrococcin GO5 production in Micrococcus sp. GO5aNitrogen sourcesb Fermentation Growth Micrococcin GO5 Specific activity time (h) (OD660 ) activity (AU ml−1 ) (AU/OD660 )None added 8 1.7 16 9.410.5% TP 8.5 5.8 128 22.071.0% TP 8 4.8 128 26.670.5% TP + 0.5% BE 8.5 6.2 128 20.650.5% TP + 1.0% BE 8 7.0 256 36.570.5% TP + 0.5% PP 8 5.8 64 11.030.5% TP + 1.0% PP 8 7.6 64 8.420.5% TP + 0.5% YE 8 5.8 128 22.070.5% TP + 1.0% YE 7 7.6 256 33.680.5% TP + 1.0% YE + 0.5% BE 7 7.6 256 33.680.5% TP + 0.5% PP + 0.5% BE 7 8.4 256 30.480.5% TP + 0.5% YE + 0.5% PP 7 7.2 128 17.780.5% TP + 0.5% YE + 0.5% BE 7 7.4 128 17.300.5% TP + 0.5% YE + 0.5% BE + 0.5% PP 7 8.2 128 15.61Data are means of duplicates. Standard errors were less than 5% of the means. a Cells were grown at 37 ◦ C in a 500 ml Erlenmeyer flask containing 50 ml of MRS medium that was modified to contain 2% lactose insteadof glucose and was supplemented with the nitrogen sources indicated instead of peptone, yeast extract, and beef extract. Cell growth, pH andbacteriocin activity were measured at time intervals, and the values when bacteriocin activity was maximum are presented. b TP, tryptone; PP, peptone; YE, yeast extract; BE, beef extract.
  • 6. M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 54–61 59Table 3Effect of inorganic phosphate concentration on growth and micrococcin GO5 production in Micrococcus sp. GO5aConcentration of Fermentation Growth Final pH Micrococcin GO5K2 HPO4 (g l−1 ) time (h) (OD660 ) activity (AU ml−1 )Not added 8 5.0 5.32 641.0 8 6.4 5.37 1282.0 8 8.0 5.41 2565.0 8 8.2 5.78 51210.0 8 6.4 5.40 102415.0 8 5.6 6.74 102420.0 7 5.4 6.83 204825.0 7 5.4 7.04 204830.0 7 4.8 7.12 512Data are means of duplicates. Standard errors were less than 5% of the means. a Cells were grown at 37 ◦ C in a 500 ml Erlenmeyer flask containing 50 ml of a modified MRS medium that contained 2% lactose, 0.5%tryptone, 1% yeast extract, 0.5% sodium acetate, 0.01% MgSO4 ·7H2 O and different concentrations of K2 HPO4 . The initial pH was adjustedto 7.0 with 5N NaOH. Cell growth, pH and bacteriocin activity were measured at time intervals, and the values when bacteriocin activity wasmaximum are presented.yeast extract were chosen as the nitrogen sources in the are consistent with a previous study by De Vuyst andcultivation medium for micrococcin GO5 production. Vandamme (1993); they reported that KH2 PO4 was the best phosphorus source for nisin production by3.5. Inorganic phosphate concentration Lactococcus lactis subsp. Lactis, that increasing the initial KH2 PO4 concentration from 0 to 5% stimulated MRS medium contains 2.0 g l−1 of dipotassium nisin biosynthesis, and that initial KH2 PO4 concentra-phosphate; therefore, the experiments to optimize the tions higher than 6% decreased nisin activity levels andinorganic phosphate concentration were conducted caused cell lysis.using the basal medium supplemented with 0.5% tryp-tone, 1.0% yeast extract and different concentrations 3.6. Magnesium sulfate concentrationof K2 HPO4 . As shown in Table 3, the increases in thefinal cell concentration corresponded to the increases In order to improve the bacteriocin yield further,in phosphate concentration up to 5.0 g l−1 ; the final cell the effect of the magnesium sulfate concentration onconcentration was highest in medium with 5.0 g l−1 growth and micrococcin GO5 production was testedof phosphate because it decreased in phosphate con- using a modified MRS medium consisting of 2% lac-centration greater than 10.0 g l−1 . The production of tose as a carbon source, 0.5% tryptone and 1% yeastmicrococcin GO5 also increased according to increases extract as nitrogen sources, 2.25% potassium phos-in the phosphate concentration up to 25.0 g l−1 . The phate, 0.5% sodium acetate and different concentra-highest bacteriocin activity was observed with medium tions of magnesium sulfate. Table 4 shows that thecontaining phosphate at 20.0–25.0 g l−1 ; the activity activity of micrococcin GO5 was enhanced by the addi-(2048 AU ml−1 ) was eight-fold that obtained at a phos- tion of magnesium sulfate and reached a maximumphate concentration of 2.0 g l−1 . Phosphate concentra- (4,096 AU ml−1 ) at MgSO4 ·7H2 O concentrations oftions greater than 30.0 g l−1 inhibited both growth and 5.0–10.0 g l−1 , while cell growth was affected little bybacteriocin production. Therefore, the optimal concen- the concentration of magnesium sulfate. The effectstration of K2 HPO4 appeared to be 22.5 g l−1 . The fact of other metal ions, such as Mn2+ , Zn2+ , Cu2+ , Ca2+that the highest bacteriocin activity was obtained at and Co2+ , on bacteriocin production were tested, buta phosphate concentration of 20.0–25.0 g l−1 which no significant effects were observed (data not shown).growth was rather inhibited indicates that the enhance- Growth in medium with an optimized compositionment of bacteriocin activity by the increase of phos- improved micrococcin GO5 production 32-fold com-phate concentration was not due to the increase of pared with that in MRS medium (4096 AU ml−1 versusgrowth by a buffering effect of phosphate. These results 128 AU ml−1 , respectively). The optimized medium
  • 7. 60 M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 54–61Table 4Effect of magnesium sulfate concentration on growth and micrococ-cin GO5 production in Micrococcus sp. GO5aMgSO4 ·7H2 O Fermentation Growth Micrococcin GO5concentration time (h) (OD660 ) activity (AU ml−1 )(g l−1 )Not added 8 6.4 5120.1 8 6.4 20480.5 8 6.4 20481.0 8 7.4 20485.0 7 6.6 40967.0 7 6.6 409610.0 7 7.0 4096Data are means of duplicates. Standard errors were less than 5% ofthe means. Fig. 4. Comparison of growth (open symbols) and micrococcin GO5 a Cells were grown at 37 ◦ C in a 500 ml Erlenmeyer flask con- production (closed symbols) profiles for Micrococcus sp. GO5 intaining 50 ml of a modified MRS medium that consisted of 2% MRS medium ( , ) and TY medium ( , ). Cells were grown inlactose, 0.5% tryptone, 1% yeast extract, 0.5% sodium acetate, 2.25% a bioreactor in 3 l of MRS or TY medium at 37 ◦ C with agitation atK2 HPO4 and different concentrations of MgSO4 ·7H2 O. Cell growth, 700 rpm and aeration at 0.5 vvm; the pH was controlled at 6.0 withpH and bacteriocin activity were measured at time intervals, and the 5N NaOH. Data points are means of duplicates. Standard errors werevalues when bacteriocin activity was maximum are presented. less than 5% of the means.consisting of the above-modified MRS medium sup- micrococcin GO5 decreased rapidly with prolongedplemented with 5.0 g l−1 of magnesium sulfate was cultivation after the stationary phase. These featuresnamed tryptone yeast (TY extract) medium and was of the bacteriocin production profile are common toused in further experiments including studies on culti- almost all lactic acid bacteria that produce bacteriocinsvation time courses in a bioreactor. (De Vuyst and Vandamme, 1994; Green et al., 1997; Hur et al., 2000). During the practical production of3.7. Growth and bacteriocin production profiles in bacteriocin, it is necessary to either stop the cultivationMRS and TY media using a bioreactor process or maintain the maximum level of bacteriocin activity by other methods. It has been suggested that Growth and bacteriocin production time courses the decrease in bacteriocin activity during the station-were determined in 3 l of MRS or TY medium in ary phase might be due to degradation by specific ora 5 l bioreactor that was maintained at 37 ◦ C, con- non-specific proteases, adsorption to the producer cells,trolled at pH 6.0, agitated at 700 rpm and aerated at and/or aggregation (De Vuyst and Vandamme, 1992;1 vvm; the results are shown in Fig. 4. The growth Joerger and Klaenhammer, 1986; Parente et al., 1994).rate and final cell concentration were higher in MRS The decrease of micrococcin GO5 after the stationarymedium than in TY medium. However, micrococcin phase could be eliminated by lowering the pH belowGO5 production in TY medium was 16-fold that in 3.0 or the temperature below 4 ◦ C, suggesting that theMRS medium. The production of micrococcin GO5 activity decrease might be mainly due to the proteolyticshows primary metabolite kinetics. The extracellular degradation of bacteriocin.bacteriocin activity increased rapidly during late loga- In a previous report (Kim et al., 2005), we describedrithmic growth phase, reached a maximum during early micrococcin GO5 as a new and novel bacteriocin pro-stationary phase (7 h), and was maintained at the max- duced by Micrococcus sp. GO5, which has a broadimum level for 1 h. The data imply that the production spectrum of antimicrobial activity and is heat- and pH-of micrococcin GO5 is closely associated with growth stable like nisin, but has a structure that differs frombut is not necessarily proportional to the growth rate that of nisin based on molecular weight (5.0 kDa),or cell concentration. Based on our data, the phosphate amino acid composition, and N-terminal amino acidconcentration seems to play an important role in the sequence. To increase the potential of micrococcinregulation of bacteriocin biosynthesis. The activity of GO5 for application as a food and feed biopreserva-
  • 8. M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 54–61 61tive, the enhancement of the bacteriocin productivity and evidence for stimulation of bacteriocin production underwas needed. Optimization of the cultivation medium unfavorable growth conditions. Microbiology 142, 817–827. Green, G., Dicks, L.M.T., Bruggeman, G., Vandamme, E.J., Chikin-and conditions, as described in this report, will make das, M.L., 1997. Pediocin PD-1, a bactericidal antimicrobialits application more feasible. Strain improvement for peptide from Pediococcus damnosus NCFB 1832. J. Microbiol.the production of micrococcin GO5 is now in progress. Biotechnol. 83, 127–132. Hur, J.W., Hyun, H.H., Pyun, Y.R., Kim, T.S., Yeo, I.H., Baik, H.D., 2000. Identification and partial characterization of lacticin BH5,Acknowledgement a bacteriocin produced by Lactococcus lactis BH5 isolated from Kimchi. J. Food Prot. 63, 1707–1712. Joerger, M.C., Klaenhammer, T.R., 1986. Characterization and This work was supported by the Hankuk University purification of helveticin J and evidence for a chromosomallyof Foreign Studies Research Fund 2003. encoded bacteriocin produced by Lactobacillus helveticus 481. J. Bacteriol. 167, 439–446. John, W.M.M., Ingrid, J.B., 1991. Identification and characteriza- tion of the lantibiotic nisin variant. Eur. J. Biochem. 201, 581–References 584. Kaiser, A.L., Montville, T.J., 1993. The influence of pH and growthBiswas, S.R., Ray, P., Johnson, M.C., Ray, B., 1991. Influence of rate on production of bacteriocin, bavaricin MN, in batch and growth conditions on the production of bacteriocin Pediocin AcH continuous culture. J. Appl. Bacteriol. 75, 536–540. by Pediococcus acidilactici H. Appl. Environ. Microbiol. 57, Kim, M.H., Kong, Y.J., Baek, H., Hyun, H.H., 2005. Purification and 1265–1267. characterization of micrococcin GO5, a bacteriocin produced byCheigh, C.I., Choi, H.J., Park, H., Kim, S.B., Kook, M.C., Kim, T.S., Micrococcus sp. GO5 isolated from kimchi. J. Food Prot. 68, Hwang, J.K., Pyun, Y.R., 2002. Influence of growth conditions on 157–163. the production of a nisin-like bacteriocin by Lactococcus lactis Matsusaki, H., Endo, N., Sonomoto, K., Ishizaki, A., 1996. Lantibi- subsp. lactis A164 isolated from kimchi. J. Biotech. 95, 225–235. otic nisin Z fermentative production by Lactococcus lactis 10-1:Choi, H.J., Cheigh, C.I., Kim, S.B., Pyun, Y.R., 2000. Production of relationship between production of the lantibiotic and lactate and a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 cell growth. Appl. Microbiol. Biotechnol. 45, 36–40. isolated from kimchi. J. Appl. Microbiol. 88, 563–571. Parente, E., Ricciardi, A., Addario, G., 1993. An assessment of opti-Daba, H., Lacroix, C., Huang, J., Simard, R.E., 1993. Influence of mal conditions for bacteriocin production by Lactococcus lactis growth conditions on production and activity of mesenterocin 140 NWC. In: Zamorani, A., Manachini, P.L., Bottazzi, V., Cop- 5 by a strain of Leuconostoc mesenteroides. Appl. Microbiol. pola, S. (Eds.), Biotechnology and Molecular Biology of Lactic Biotechnol. 39, 166–173. Acid Bacteria for the Improvement of Foods and Feeds Qual-Delves-Broughton, J., 1990. Nisin and its uses as a food preservative. ity. Instituto Poligrafico e Zecca dello Stato, Rome, pp. 328– Food Technol. 44, 100–117. 334.De Vuyst, L., Vandamme, E.J., 1992. Influence of the carbon source Parente, E., Ricciardi, A., Addario, G., 1994. Influence of pH on on nisin production in Lactococcus lactis subsp. lactis batch fer- growth and bacteriocin production by Lactococcus lactis subsp. mentations. J. Gen. Microbiol. 138, 571–578. lactis 140NWC during batch fermentation. Appl. Microbiol.De Vuyst, L., Vandamme, E.J., 1993. Influence of the phosphorus Biotechnol. 41, 388–394. and nitrogen source on nisin production in Lactococcus lactis Parente, E., Hill, C., 1992. A comparison of factors affecting the subsp. lactis batch fermentations using a complex medium. Appl. production of two bacteriocins from lactic acid bacteria. J. Appl. Microbiol. Biotechnol. 40, 17–22. Bacteriol. 73, 290–298.De Vuyst, L., Vandamme, E.J., 1994. Antimicrobial potential of Tagg, J.R., Dajani, A.S., Wannamaker, L.W., 1976. Bacteriocins of lactic acid bacteria. In: De Vuyst, L., Vandamme, E.J. (Eds.), Gram positive bacteria. Bantered. Rev. 40, 722–756. Bacteriocins of Lactic Acid Bacteria. Blackie Academic and Pro- Verellen, T.L.J., Bruggeman, G., Van Reenen, C.A., Dicks, L.M.T., fessional, Glasgow, pp. 91–142. Vandamme, E.J., 1998. Fermentation optimization of PlantaricinDe Vuyst, L., Callewaert, R., Crabbe, K., 1996. Primary metabolite 423, a bacteriocin produced by Lactobacillus plantarum 423. J. kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus Ferm. Bioeng. 86, 174–179.