Novel plantaricin UG1 production by Lactobacillus plantarum UG1 in enriched whey permeate in batch fermentation processes Gamal Enan 1* and Abdul Aziz Al Amri
2
Laboratory of Applied Bacteriology, Botany Department, Faculty of Science, Zagazig University, Zagazig, Egypt. 2 Department of Sciences, King Khalid Military Academy, Box 22140, Riyadh 11495, Saudi Arabia. *e-mail: gamalenan 610 @ hotmail. com,
[email protected]
1
Received 5 January 2006, accepted 29 March 2006.
Abstract Lactobacillus plantarum UG1 grew in whey permeate (WP) and produced the bacteriocin plantaricin UG1, although the biomass and bacteriocin titres were lower than those obtained in the control MRS medium. Upon enrichment of WP by yeast extract and some mineral salts, L. plantarum UG1 gave comparable values of growth and plantaricin UG1 activity. The enriched WP medium was designated whey permeate–yeast extract–mineral salts (WYM).The composition of this medium is indicated in the text. The dynamics of plantaricinUG1 biosynthesis by L. plantarum UG1 were studied in WYM medium in batch fermentation processes. Maximum plantaricin UG1 activity was obtained in WYM medium when the producer organism was in the late exponential and in the early stationary phase of growth. Titres of plantaricin UG1 in WYM medium were almost 9% higher in pH-controlled than in pHuncontrolled fermentations. Key words: Lactobacillus plantarum UG1, bacteriocin/plantaricin UG1, whey permeate/fermentation.
Introduction Bacteriocins are mainly heterogenous molecules (major component is protein) possessing broad spectrum of activity including spoilage bacteria and food–borne pathogens, different mechanisms of action and complicated chemical nature 10, 20. Bacteriocins of lactic acid bacteria have attracted the attention of many investigators especially in the recent years because of their use as natural preservative in food industry with increasing safety and for their role in extending the shelf life of foods 12. The bacteriocin plantaricin UG1 employed in this study, was produced by Lactobacillus plantarum UG1 (L. plantarum UG1). It was inhibitory to many food–borne pathogens and thermostable and active over wide pH range ( pH 4-7) 11. It was produced in both complex and synthetic media in batch fermentation processes 1. It is of interest to use WP as a culture medium for bacteriocin production. This is due to its cheap cost. WP is a by-product of cheese industry. Approximately, 1 kg of whey results for every kilogram of cheese produced and the cost associated with disposing this large volume of whey is substantial 24. Furthermore, the high chemical oxygen demand (COD) (O2 50 kg/ton) of whey makes its disposal a pollution problem 15. Therefore, use of whey as fermentation feedstock has long been of industrial interest. Bacteriocin (nisin, pediocin AcH) production in WP was reported in previous studies 6. However, nothing was published on plantaricins production by L. plantarum in WP. To fill this gap, the goals of this study were to (a) produce plantaricin UG1 by L. plantarum UG1 in supplemented WP, (b) optimize plantaricin UG1 production in supplemented WP in batch fermentation processes and (c) study the dynamics of plantaricin UG1
formation in pH–uncontrolled and pH-controlled batch fermentations. Materials and Methods Bacterial strains: L. plantarum UG1, the producer of plantaricin UG1, was isolated, characterized and identified as described previously 10. This UG1 strain was maintained as frozen stocks at – 20oC in MRS broth plus 10% glycerol and was propagated in MRS broth 5, 14. Lactococcus lactis MG1614 was used as the indicator organism. It was produced from Laboratory of Food Technology and Nutrition, Faculty of Agricultural & Applied Biological Sciences, University of Gent, Belgium. It was maintained at – 20oC in skimmed milk (Difco) and was propagated every two weeks in brain heart infusion broth (BHI, Difco). Determination of the bacterial dry weight: Growth was measured as cell dry weight (CDW, g/l ) determination. Culture broths were centrifuged at 10,000 x g for 10 min in preweighed tubes. The pellets were washed with distal water and dried at 105oC for 24 h to measure cell dry weight. Preparation of the inoculum: L. plantarum UG1 preculture was grown for 10 h at 30oC in 10 ml MRS broth. The organism was subcultured for 12 h at 30oC in 50 ml MRS broth. A broth culture of actively exponentially growing cells was used as an inoculum. The inoculum size was always 1% v/v (1.5x108 cfu/ml). Plantaricin UG1 production and bioassay: L . plantarum UG1, the producer of plantaricin UG1, was grown in liquid broth under
test for 16 h at 30oC..Cell–free supernatant was obtained by centrifuging the culture at 10,000 x g for 10 min at 4oC. The pH of the cell free supernatant was then adjusted to 6.5 to exclude the inhibitory activity due to organic acids. This pH-adjusted cell free supernatant was filter–sterilized through cellulose membrane filters (Amicon, 0.45 µm Millipore). Quantitative estimation of the antibacterial titres of the pH-adjusted, filter-sterilized cell free supernatant was carried out by the critical dilution assay 23. Plantaricin UG1 production in enriched WP: Whey was provided from Nadec Dairy Company (Saudi Arabia) and was heated at 121oC for 15 min to denature proteins. The precipitated proteins were eliminated by filtration on cheese cloth. The permeate was collected and its initial pH was adjusted at 6.5. It was then autoclaved at 121oC for 20 min and used as a culture medium. The effects of supplements were tested in 500 ml Erlenmeyer flasks, each containing 250 ml broth medium adjusted initially to pH 6.5 (the optimum pH value for plantarin UG1 production) 10. The supplements tested and their concentrations are listed in Table 1. The experimental samples were inoculated by L. plantarum UG1, the bacteriocin producer strain, incubated at 30oC for 16 h and were then analyzed for growth values (CDW g/l ), medium acidity (final pH) and plantaricin UG1 activity as described above. Bioreactor startup: The bioreactor used was 2 L bench–top fermentor with a flat–bottom stirred vessel (Applikon, Scheidam, Netherlands) connected to pH–controller (Parmer, Illinois, USA). A muster flex pump with fixed speed drive 220 VAC pumphead with CRS rotor was connected to pH–controller for automatic addition of 1 M NaOH to keep the pH at 6.5 in pH-controlled fermentations. Dynamics of plantaricin UG1 formation in pH-uncontrolled and pH–controlled fermentations: The flat-bottom tank bioreactor was filled with 1 litre of whey permeate–yeast extract–mineral salts medium (WYM medium) and was then autoclaved at 121oC for 20 min. To study the effect of agitation, the bioreactor experimental WYM medium was inoculated with 1% v/v L. plantarum UG1 cells. The experiment was performed one time without agitation and was carried out also at agitation values of about 50–300 rpm (Table 2). To construct the batch fermentation profiles, the fermentor was operated at 30oC with 100 rpm (the optimum agitation value) and with controlled pH (at pH 6.5) or without pH controller. During the fermentation period, samples (10 ml) were aseptically withdrawn at regular time intervals and analyzed for growth (CDW g/l) and plantaricin UG1 activity (AU/ ml) 1. Results and Discussion The conditions of the bacteriocin plantaricin UG1 production used in this study were optimized in a previous work 8-10. In the current investigation, L. plantarum UG1grew in WP and produced plantaricin UG1 (Table 1), although the biomass and bacteriocin activity were lower than those obtained in the control MRS medium and were also less than those obtained when WP was enriched (Table 1). Few publications also reported the feasibility of the production of both nisin and pediocin AcH bacteriocins in WP 17. Little information is available concerning growth of Lactobacillus plantarum species in dairy products, although the growth of L. plantarum species in some dairy products was reported 7. Also
Table 1. Influence of culture media on final pH, growth (Cell dry weight, CDW g/l) and plantaricin UG1 activity after 16 h of growth of Lactobacillus plantarum UG1 at 30°C. Culture media tested
CDW (g/l )
Final pH
Control medium (MRS)* Whey permeate (WP) only Whey permeate (WP) supplementation (a) WP + growth factor (yeast extract) 0.5% ( %W/V) 1.0 % 1.5%+ 2.5% (b) WP + 1.5% yeast extract + 0.2% of K3PO4 of K2HPO4 of K H2PO4 of Na3 PO4 of Na2 HPO4 of NaH2PO4 ©WP + 1.5% yeast extract + 0.2% K2HPO4 + either of…(g/l)… Ammonium citrate (0.2)+ Amm. nitrate (0.2) Sodium acetate (0.2) Sodium nitrate (0.2) MnSO4 (0.01) (Amm. citrate–Na acetate +MnSO4)+
2.21 1.5
3.94 4.52
Plantaricin UG1 activity (AU/ml) 5780 2580
1.9 2.0 2.11 2.11
4.4 4.2 4.0 4.0
3800 3820 4280 4220
2.0 2.20 2.0 1.8 1.8 1.9
3.8 3.96 3.7 4.2 4.5 4.6
4200 4320 4200 3200 3200 3200
2.11 1.8 1.7 1.7 1.8 2.2
4.0 4.4 4.3 4.2 4.6 3.82
5600 5000 5000 4800 5100 5880
+Optimum concentration for growth and bacteriocin production by L.plantarum UGI * MRS (DeMan, Rogosa and Sharpe broth 1960).
Table 2. Effect of agitation on growth (Cell dry weight, CDW g/l) , final pH and plantaricin UG1 activity after 16 h of growth of Lactobacillus plantarum UG1 at 30°C. Agitation in round per minute (rpm) 0 50 100 * 150 200 250 300
CDW (g/l )
Final pH
2.21 2.18 2.24 2.20 2.22 2.24 2.26
3.82 3.60 3.86 4.00 3.98 8.96 3.96
Plantaricin UG1 activity (AU/ml) 5880 5900 6200 5600 5440 4880 5000
*The optimum agitation condition for bacteriocin production by L. plantarum UG1.
the growth of L. plantarum UG1 in pasteurized milk and WP was reported 12. The growth and bacteriocin production ability of lactic acid bacteria were shown to be correlated 20. Hence, growth and plantaricin UG1 production ability in WP were detected in this work. This was possible because WP contains 4.9% lactose, 0.03% protein, 0.1% non-protein nitrogen, 0.15% lactic acid and less than 0.01% fat 3. The lactose in whey permeate is a suitable carbon source for many microorganisms. In addition WP is rich in minerals and contains vitamins which may provide valuable nutrients to stimulate bacterial growth and bacteriocin production. By enrichment of WP with different concentrations of yeast extract, plantaricin UG1 production was improved. The results presented in Table 1 indicate that 1.5% yeast extract supplementation yielded 39.72% more plantaricin UG1 than in WP only. Yeast extract has also been reported to increase the production of bacteriocins, namely mesenterocin 5 4, pediocin AcH and nisin 24. WP containing 1.5% yeast extract was subjected
2.5
aa
CDW(g/l)
2 1.5 1 0.5 0
0
10
20
30
40
50
60
40
50
60
40
50
60
Plantaricin UG1 activity AU/ml
Time(h)
7000
b
6000 5000 4000 3000 2000 1000 0 0
10
20
30 Time(h)
7
c
Final pH
6 5 4 3 2 1 0
0
10
20
30 Time(h)
Figure 1. Batch fermentation profiles of L. plantarum UG1 growth and plantaricin UG1 production in WYM medium at uncontrolled pH value. Cell dry weight (CDW,g/l) (a) ; plantaricin UG1 activity (AU/ml) (b) ; final pH value (c).
Plantaricin UG1 activity AU/ml
8000 7000 6000 5000 4000 3000 2000 1000 0
0
10
20
30 Time(h)
40
50
60
Figure 2. Batch fermentation profiles of L. plantarum UG1 plantaricin UG1 production (plantaricin UG1 activity, AU/ml) in WYM medium at controlled pH value (pH 6.5).
to mineral salt supplementations. Results are given in Table 1. K2HPO4, ammonium citrate and MnSO4 improved bacteriocin production by L. plantarum UG1. A noticeable increase of bacteriocin production from 4280 to 5880 AU/ml was observed upon medium supplementation (wp + 1.5% yeast extract) with mixture of K2HPO4, ammonium citrate, sodium acetate and MnSO4 (Table 1). Hence, a medium designated WP-yeast extract–mineral salts (WYM) was observed to maximize plantaricin UG1 production by L. plantarum UG1. The composition of this medium was: 1 litre WP, 1.5% yeast extract, 0.2% potassium monohydrogen phosphate, 0.02% ammonium citrate, 0.02% sodium acetate, 0.01% manganese sulphate. Values of plantaricin UG1 and growth in WYM medium were almost comparable to those obtained in the Lactobacillus MRS medium. The mineral salts were known to increase bacteriocin production by lactic acid bacteria. K2HPO4 contains phosphorus source which is very important for bacteriocin production 16. MnSO 4 doubled lactostrepcin activity 18. Moreover, enrichment of TGE medium with citrate and Mn or Mg acetate resulted in an optimal pediocin AcH production by Pediococcus acidilactici 2. The surfactant Tween 80 has no effect (data not shown) on bacteriocin production by L. plantarum UGL. This result supported previous results in this respect 1, 10. The effect of agitation on L. plantarum UG1 growth and plantaricin UG1 production is shown in Table 2. The agitation value of about 100 rpm was the best for L. plantarum UG1 growth and plantaricin UG1 production (Table 2). The antibacterial titres increased from 5880 AU/ml in static culture to 6200 AU/ml in agitated culture (at 100 rpm ). This result is in conform with many previous findings 19, 24 . The dynamics of plantaricin UG1 formation in WYM medium were studied in batch fermentation processes. The results are given in Fig. 1 and 2. It is evident that the exponential growth of L. plantarum UG1 started during 5–8 hours in both pHuncontrolled (Fig. 1) and pH–controlled (Fig. 2) fermentation. Plantaricin UG1production was not detected in the early exponential phase, but it was detected after 8 h of fermentations in both cases (with or without pH-control) in the mid logarithmic phase of the producer organism (Fig. 1 and 2). Maximum plantaricin UG1 production of 6400 AU/ml (Fig. 1) and 7000 AU/ml (Fig. 2) was obtained after 16 h in pH–uncontrolled and pH-controlled fermentations, respectively, when the producer organism was in the late exponential and in the early stationary phase of growth. The growth of L. plantarum UG1 cells correlated with bacteriocin production. The maximum plantaricin UG1 production was almost 9% higher in pH–controlled than in pH-uncontrolled fermentation. Plantaricin UG1 production declined particularly in pHuncontrolled fermentations with a prolonged fermentation time (Fig. 2). The decrease in plantaricin UG1 activity at later stages of growth may be due to adsorption of bacteriocin to live and dead cells of the producing organism 13, 22. The sharp decrease in plantaricin UG1titres after 16 h in pH-uncontrolled fermentations may be due to the effect of acidic pH values on the stability of this bacteriocin 8-10. Similar fermentation patterns were obtained for several lactic acid bacteria producing bacteriocins 21. The slight decrease of plantaricin UG1 in pH-controlled fermentation in the stationary phase of growth of the producer organism implies that the fermentation processes would need to
be stopped to achieve maximum plantaricin UG1 production. This was also proposed for enterocin 1146 21. The production of plantaricin UG1 by high titres in WYM medium showed a quite promising interest to use the pollutant agricultural by–product whey permeate in a useful applications. The fermented WP by L. plantarum UG1, the producer of plantaricin UG1, can be used as natural biopreservative in many food products. The work in this respect is in progress. Acknowledgement The authors are indebted to King Khalid Military Academy for allowing us to carry out this work. Dr. Gamal Enan is indebted to Prof. Dr. Ir.J Debevere,the Chairman of Department of Food Technology and Food Preservation,University of Gent, Belgium for providing the experimental strains.
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