PRODUCTION OF DRY DIVERCIN PREPARATIONS BY SPRAY DRYING AND LYOPHILIZATION

Radosław Dembczyński¹, Piotr Lubbe¹, Wojciech Białas¹, Anna Sip¹, Tomasz Jankowski¹, Włodzimierz Grajek², Roman Marecik¹, Paweł Cyplik^1
1 Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poland
² Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poznań, Poland

ABSTRACT

Divercin preparations were obtained in the process of freeze and spray drying where high-saccharified maltodextrin was used as a drying carrier. It was found that spray drying of a liquid divercin extract was possible when maltodextrin at a concentration of at least 10% was applied while a liquid divercin extract could be freeze-dried without the addition of maltodextrin. However, when the concentration of maltodextrin in the dried liquid extracts was lower than 10%, freeze-dried divercin preparations contained more than 10% of water and could be microbiologically unstable and susceptible to non-enzymatic transformations. It was also found that freeze drying and spray drying processes did not decrease divercin activity.

Key words: bacteriocins, divercin, Listeria, spray drying, freeze drying.

INTRODUCTION

Divercin is a bacteriocin which exhibits antibacterial activity against pathogenic Listeria species [15]. At present these bacteria are becoming a great problem because they can cause serious illnesses when eaten with contaminated food products. Listeria has the ability to grow at low temperatures and is frequently found in food products stored in the refrigerators [5,6,8,9]. Divercin added to foods alone or in combination with traditional and modern preservation techniques can eliminate foodborne pathogens and enhance the safety and shelf life of foods [2,16]. Divercin can also be used as a constituent of disinfectants for food industry.

Divercin is produced by a lactic acid bacterium Carnobacterium divergens. During cultivation this bacteriocin is exogenously excreted to the medium. A liquid divercin extract is obtained after biomass separation [14].

Drying of this extract makes it possible to produce easy-to-use, microbiologically stable divercin preparations and conserve bacteriocin activity for longer periods of time at room temperatures. In the present work dry preparations of divercin were obtained by freeze and spray drying of divercin extracts with maltodextrin as a drying carrier. Also assessed was the influence on divercin activity of the technological processes applied.

MATERIAL AND METHODS

Microorganisms and culture conditions
Carnobacterium divergens AS7, a lactic acid bacteria strain from the departmental collection, previously isolated from the salmon digestive tract has been used. Batch cultures were performed in quasi-anaerobic conditions in a 5 dm³ Bioflo III fermentor (New Brunswick Sci. Edison, N.J., USA) at 30°C and a stirring speed of 100 rpm.

The bacteria were grown on a whey medium. Sweet cheese whey powder (Lactopol, Białystok, Poland) in the amount of 30.85 g l^-1 was dissolved in a solution containing salts and Tween 80 (2g l^-1 K₂HPO₄, 2 g l^-1 (NH₄)₂C₆H₆O₇, 5 g l^-1 CH₃CO₂Na·3H₂O, 0.57 g l^-1 MgSO₄·7H₂O, 0.14 g l^-1 MnSO₄·5H₂O, 1g l^-1 Tween 80). The pH of the medium was adjusted to 4.5 by titration with H₃PO_4. After autoclaving at 121°C for an hour, the precipitates were removed by continuous centrifugation at 12 000 g (Z 61, Cepa, Scharzwald, Germany). After the second sterilization (121^oC, 30 min), the pH of the medium was adjusted to 6.5 with 5 M NaOH.

A fresh medium at a working volume of 4 dm³ was inoculated with 2% (v/v) of an exponentially growing culture following two transfers into the MRS broth (BTL, Łódź, Poland). The pH of the culture was kept constant at 6.5 by automatic addition of 5 M NaOH. Cultures were performed in triplicate.

Fermentations were ceased in the late logarithmic phase. The cells of Carnobacterium divergens were removed from the medium by centrifugation at 3800 g (15 min, 4°C, Multifuge 3 S-R, Heraeus, Germany). After supernatant heating at 80°C for 15 min to inactivate proteolytic enzymes, the divercin extract was obtained.

Indicator bacteria, Listeria innocua F (E.N.I.T.I.A.A. Nantes, France), used for the determination of divercin activity, has been cultured at 30°C in a medium containing 1% glucose, 1% sodium chloride and 0.5% yeast extract.

Freeze drying and spray drying process
Prior to drying, liquid divercin extracts were mixed with drying carrier, which was high-saccharified maltodextrin (DE25, Nowamyl, Łobez, Poland). The concentration of maltodextrin in divercin extracts ranged from 5 to 30% with increments of 5%. Samples without addition of maltodextrin were freeze- and spray-dried, too.

In the case of freeze drying, samples were frozen at –35°C in a freeze-dryer chamber (Beta 1-16, Christ, Germany). Main drying cycle was performed at 15°C for 15 h at a pressure of 22 Pa. Final drying was carried out at a shelf temperature of 20°C for two hours.

The spray drying process of divercin extracts was carried out in a Mobile Minor 2000 (Niro A/S, Denmark) spray dryer equipped with a co-current two-fluid nozzle system with pneumatic atomization. The inlet air temperature was set at 180°C and the outlet air temperature was maintained at 80°C by adjusting the flow rate of the feed solution.

Antibacterial activity assay
The activity of divercin was determined immediately after drying by the method of critical dilutions, described by Pilet et al. [11] using Listeria innocua F as target organism in triplicate. Antibacterial activity was expressed as Activity Units (AU ml^-1), which denote the lowest concentration of a sample which does not exhibit the ability to inhibit growth of indicator bacteria. The activity of divercin was tested in divercin extracts before drying and in dry divercin preparations. In the latter case solutions with the same concentration of solids as in the liquids before drying were prepared prior to activity assay.

Determination of dry matter
Solid content in the liquid divercin extracts and dry divercin preparations was monitored gravimetrically after drying to a constant weight at 105°C. All samples were analyzed in triplicate.

RESULTS AND DISCUSSION

Bacteriocins of lactic acid bacteria are considered natural biopreservatives since lactic acid bacteria are regarded as GRAS microorganisms and bacteriocins are degraded by proteases in the gastrointestinal tract [7]. The application of bacteriocins as food preservatives can be achieved by using bacteriocins as food additives. However, the usefulness of liquid bacteriocin preparations can be questionable. High water content in liquid bacteriocin preparations is the main cause of their instability in terms of preserving antimicrobial activity during prolonged storage. Liquid preparations also have low activity per unit volume. Thus, the application of dry bacteriocin preparations instead of liquid ones is more desirable. Dry powders can be transported at a low cost and can be stored in a stable form for long periods of time [4]. Thus, the aim of the present study was to obtain dry divercin preparations by means of freeze and spray drying.

From a commercial point of view an inexpensive method of large-scale production of dry bacteriocin preparations is highly desirable. However, during the dewatering of protein solutions by drying some adverse processes such as protein denaturation and the loss of biological activity can occur [17]. Hence, in this study the changes in divercin activity during drying were monitored. The experiments conducted showed a high thermal and desiccation stability of divercin. It was found that both freeze and spray drying did not negatively influence the divercin activity (Fig. 1). Similar results were obtained by other authors who dealt with freeze and spray drying of different bacteriocins [3,10,13].

Fig.1. Changes of divercin activity during freeze and spray drying

Another advantage of dry divercin preparations over liquid extracts was their higher activity per mass unit (Fig. 2). The highest divercin activity of 9475±4297 AU g^-1 was achieved in powders obtained by freeze drying of bacteriocin extracts without the addition of maltodextrin. However, the moisture content in these preparations was close 25% (Table 1). Water content over 10% was recorded for divercin preparations obtained by freeze drying of liquid extracts with the addition of maltodextrin at a concentration of 5%. Because of high moisture content these preparations could be microbiologically unstable. Other samples, not mentioned here, mostly had the moisture content of about 4-5%, which should minimize the risk of microbial deterioration during storage.

Fig.2. Divercin activity in liquid extracts before drying and in freeze- and spray-dried preparations

Table. 1. Solid contents in liquid divercin extracts and dry divercin preparations

Maltodextrin concentration in liquid extract (%)	Solid content (%)
	liquid extract	dry preparations
		freeze-dried	spray-dried
0	3.42±0.44	76.24±3.37	-
5	8.32±0.76	89.49±0.89	-
10	12.93±1.06	92.65±0.26	95.84±0.88
15	16.28±0.92	94.71±0.56	96.45±0.20
20	19.54±0.66	95.04±0.52	96.66±0.18
25	25.88±0.84	95.22±0.68	96.74±0.07
30	30.19±1.33	95.48±0.54	96.85±0.09

It was found that divercin extracts should contain at least 10% of maltodextrin for successful spray drying. Otherwise, extracts stuck to the walls of the drying chamber. The stickiness observed is attributed to low molecular weight sugars and organic acids in the dried product [1]. In the present study, lactic acid produced by bacteria during fermentation was probably responsible for the stickiness. This problem was solved by the addition of an appropriate amount of maltodextrin to the divercin extract. Indeed, the stickiness was not present any more for a maltodexrin concentration of 10% or higher in the spray-dried divercin extracts.

Freeze drying costs are 4-8 times higher as compared to air drying [12]. In this work, dry samples obtained by freeze and spray drying showed comparable divercin activity. For instance, when maltodextrin at a concentration of 10% was added to the divercin extract, the bacteriocin activity achieved was 3090±1338 and 3202±1408 AU g^-1 after freeze and spray drying respectively. Thus, in the light of the results presented in this study, the spray drying should be recommended as a method of production of dry divercin preparations.

CONCLUSIONS

There is no difference in the quality of the dry divercin preparations obtained by freeze and spray drying. Thus, for the economical reasons spray drying should be recommended as a preferred manufacturing method of dry divercin preparations. However, for the successful spray drying the maltodextrin in the concentration of 10% should be added to the divercin liquid extract.

ACKNOWLEDGMENTS

The research presented in this paper was financially supported by the Polish Committee for Scientific Research (project No PBZ-MIN-007/P04/2003).

REFERENCES

1. Adhikari B., Howes T., Bhandari B.R., Troung V., 2004. Effect of addition of maltodextrin on drying kinetics and stickiness of sugar and acid-rich foods during convective drying: experiments and modeling. J. Food Eng. 62, 53-68.

2. Bhugaloo-Vial P., Grajek W., Dousset X., Boyaval P., 1997. Continuous bacteriocin production with high cell density bioreactors. Enzyme Microb. Technol. 21, 450-457.

3. Dimitrieva-Moats G., Yüksel. G.Ü., 2005. Prospective use of freeze-dried preparations of bacteriocins from lactic acid bacteria to inhibit Listeria monocytogenes and Staphylococcus aureus in dairy products. IFT Annual Meeting, July 15-20 - New Orleans, Louisiana. Food Microbiology: Antimicrobial effects on foodborne microorganisms, 89D-26.

4. Gardiner G.E., O’Sullivan E., Kelly Y., Auty M.A.E., Fitzderald G.F., Collins Y. K., Ross R.P., Stanton C., 2001. Comparative survival rate of human-derived probiotic Lactobacillus paracasei and L. salivarius strains during heat treatment and spray-drying. Appl. Environ. Microbiol. 66, 2605-2612.

5. Harvey J., Glimour A., 2001. Characterization of recurrent and sporadic Listeria monocytogenes isolates from raw milk and nondairy foods by pulsed-field gel electrophoresis, monocin typing, plasmid profiling, and cadmium and antibiotic resistance determination. Appl. Environ. Microbiol. 67, 840-847.

6. Hassan Z., Purwati E., Radu S., Rahim R.A., Rusul G., 2001. Prevalence of Listeria sp. and Listeria monocytogenes in meat and fermented fish in Malaysia. Southeast. Asian J. Trop. Med. 32, 402-407.

7. Holtzapfel W. H., Geisen R., Schillinger U., 1995. Biological preservation of foods reference to protective cultures, bacteriocins and food-grade enzymes. Int. J. Food Microbiol. 24, 343-362.

8. Jorgensen L.V., Huss H.H., 1998. Prevalence and growth of Listeria monocytogenes in naturally contaminated seafood. Int. J. Food Microbiol. 30, 127-131.

9. Lin CH.-M., Fernando S.Y., Wie CH., 1996. Occurrence of Listeria monocytogenes, Salmonella sp., Escherichia coli and E. coli O157:H7 in vegetable salads. Food Control 3, 135-140.

10. Mauriello G., Aponte M., Andolfi R., Moschetti G., Villani F., 1999. Spray-drying of bacteriocin-producing lactic acid bacteria. J. Food Prot. 62, 773-777.

11. Pilet M. F., Dusset X., Barre R., Novel G., Desmazaeud M., Piard J. CH., 1995. Evidence for two bacteriocins produced by Carnobacterium piscicola and Carnobacterium divergens isolated from fish and active against Listeria monocytogenes. J. Food Prot. 58, 256-262.

12. Ratti C., 2001. Hot air and freeze-drying of high-value foods: a review. J. Food Eng. 49, 311-319.

13. Ross R.P., Galvin M., Mcauliffe O., Morgan S.M., Ryan M.P., Twomey D.P., Meaney W.J., Hill C., 1999. Developing applications for lactococcal bacteriocins. Antonie van Leeuwenhoek 76, 337-346.

14. Sip A., Grajek W., 2001. Transfer of divercin through ultrafiltration membranes after treatment with detergents and organic solvents. EJPAU 4(2), #08, http://www.ejpau.media.pl/volume4/issue2/food/art-08.html.

15. Sip A., Grajek W., 2003. Biosynthesis of divercin by Carnobacterium divergens AS7 in continuous high cell density cultures. EJPAU 6(1), #02, http://www.ejpau.media.pl/volume6/issue1/biotechnology/art-02.html.

16. Sip A., Grajek W., 2005. Activity of bacteriocin produced by Carnobacterium divergens AS7 in ground beef. Folia Univ. Agric., Sci. Aliment. 246 (4), 255-266.

17. Strumiłło C., Kamiński W., Zbiciński I., 1995. Some aspects of the drying of protein products. Chem. Eng. J. 58, 197-204.

 

Accepted for print: 12.03.2008

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Radosław Dembczyński
Department of Biotechnology and Food Microbiology,
Poznań University of Life Sciences, Poland
Wojska Polskiego 48, 60-627 Poznań, Poland
phone: +48 61 8466026
email: rdembcz@au.poznan.pl

Piotr Lubbe
Department of Biotechnology and Food Microbiology,
Poznań University of Life Sciences, Poland
Wojska Polskiego 48, 60-627 Poznań, Poland

Wojciech Białas
Department of Biotechnology and Food Microbiology,
Poznań University of Life Sciences, Poland
Wojska Polskiego 48, 60-627 Poznań, Poland
email: wbialas@au.poznan.pl

Anna Sip
Department of Biotechnology and Food Microbiology,
Poznań University of Life Sciences, Poland
Wojska Polskiego 48, 60-627 Poznań, Poland
Phone: +48 061 846 60 04

Tomasz Jankowski
Department of Biotechnology and Food Microbiology,
Poznań University of Life Sciences, Poland
Wojska Polskiego 48, 60-627 Poznań, Poland
Phone: 061 846 60 04

Włodzimierz Grajek
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poznań, Poland
ul. Wojska Polskiego 48
60-627 Poznań
Poland

Roman Marecik
Department of Biotechnology and Food Microbiology,
Poznań University of Life Sciences, Poland
Wojska Polskiego 48, 60-627 Poznań, Poland

Paweł Cyplik
Department of Biotechnology and Food Microbiology,
Poznań University of Life Sciences, Poland
Wojska Polskiego 48, 60-627 Poznań, Poland

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