Decarboxylase activity of Serratia marcescens depending on ph and chosen monosaccharide content

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1 Decarboxylase activity of Serratia marcescens depending on ph and chosen monosaccharide content Z. LAZÁRKOVÁ a), A. ANDRESOVÁ a), P. PLEVA b), E. LORENCOVÁ b), L. BUŇKOVÁ b), F. BUŇKA c) a) Department of Food Analysis and Chemistry b) Department of Fat, Tenside and Cosmetics Technology c) Department of Food Technology and Microbiology Tomas Bata University in Zlin nam. T. G. Masaryka 275, Zlin CZECH REPUBLIC Abstract: The effect of ph (6, 7 and 8) and glucose, galactose and fructose content (0.00, 0.25, 0.50, 0.75 and 1.00% w/v) on growth and decarboxylase activity of Serratia marcescens was evaluated. Nutrient broth with 0.2% w/v of ornithine was used for the cultivation of bacteria. Biogenic amine production was determined using the ion-exchange chromatography with post-column ninhydrine derivatization and spectrophotometric detection. While the optimal ph for bacteria growth was 7 8, the decarboxylases showed maximal activity at ph 6. Monosaccharides stimulated both bacteria growth and putrescine formation at ph 6. On the contrary, addition of all monosaccharides tested caused inhibition of putrescine production at ph 7 and 8. Key-Words: Serratia marcescens, ph, monosaccharides, decarboxylase activity, biogenic amines, ornithine, putrescine 1 Introduction Microorganisms are used for the production of quite a number of foods. However, their metabolic activity can affect the food quality. Microbial decarboxylases are lyases which catalyses free amino acid decarboxylation and thus biogenic amine production [1]. Likewise Serratia marcescens belong to the bacteria with decarboxylase activity [2,3,4]. S. marcescens is facultative anaerobic gramnegative bacteria from the Enterobacteriaceae family. It produces red spots thus causing the nonacid food spoilage [5]. S. marcescens is lysine and ornithine decarboxylase-positive. Cadaverine is produced from lysine owing to lysine decarboxylase (EC ) and putrescine is formed from ornithine by the action of ornithine decarboxylase (EC ) [6,7]. Decarboxylase activity can be influenced by medium composition, microorganism growth phase and conditions suitable for the decarboxylase production (fermentable saccharides and NaCl content, temperature, aero/anaerobiosis, ph, redox potential, presence of biogenic amine in medium, etc.) [2,3,8]. Biogenic amines are low-molecular organic compounds with biological activity. While the low concentrations of biogenic amines observe important function in human organism (structural, metabolic, regulating, secretive, stimulative, etc.), high amounts can lead to alimentary intoxications. Providing biogenic amines have the access to human blood system, harmful effects could be expected. There are two main physiological effects of biogenic amines psychoactive and vasoactive [3,9,10]. Biogenic amines are generally presented in almost all food as the common metabolism products. Higher levels are included in fermented foods such as cheese, sausage, wine and beer as a result of microbial activity. On the other hand, contaminant microflora produces biogenic amines mainly in meat and fish during the storage. Particularly rich in biogenic amines are foods in advanced level of spoilage [11]. Biogenic amines are not only toxic to sensitive individuals but also can have synergic effect. Thus it is important for producers to optimize and strictly follow conditions of good manufactory practice, production technology and storage requirements. Furthermore, it is necessary to use short fermentation and carefully chosen starter cultures to ensure low levels of biogenic amines in fermented food. Biogenic amine concentration is one of the food quality indicators. Hence it is fundamental to monitor biogenic amine amount in food and observe all factors influencing their production [2,3,11,12]. ISBN:

2 Last but not least, advanced detection of biogenic amines producing bacteria is essential. The aim of this study was to monitor the effect of ph (6, 7 and 8) and glucose, galactose and fructose addition ( %) on Serratia marcescens growth and decarboxylase activity (putrescine production). 2 Experimental The impact of selected factors affecting biogenic amine production was tested in Serratia marcescens CCM 303. Cultivation of bacteria was performed in Nutrient Broth (HiMedia, Bombai, India) with the addition of 0.2% (w/v) ornithine (Sigma-Aldrich, St. Louis, USA) at 30±1 C for 20 hours. For the determination of ph and monosaccharide addition effect, S. marcescens CCM 303 was cultivated in Nutrient Broth enriched by 0.2% (w/v) ornithine at 30±1 C for 20 hours. Glucose, galactose and fructose (Penta, Prague, Czech Republic) in the amount of 0.00, 0.25, 0.50, 0.75 and 1.00% (w/v) were added to culture medium. Cultivation was accomplished at ph 6, 7 and 8 (ph was adjusted by 0.1 mol/l HCl and NaOH). The cells were removed by centrifugation (15000 g for 10 min) after the cultivation of bacteria and the supernatant was filtered through a 0.45 µm filter. Putrescine production was evaluated by ion exchange chromatography (Amino acid analyser AAA400, Ingos, Prague, Czech Republic) with ninhydrine derivatization and spectrophotometric detection according to [13]. All the reagents were obtained from Ingos, Prague, Czech Republic, except for putrescine standard which was purchased at Sigma-Aldrich, St. Louis, USA. Each combination (ph/monosaccharide) was analysed six times (3 replicate tubes and 2 analyses in AAA). Besides the putrescine production the following factors were evaluated: (i) ph of the medium after the cultivation (ph-meter CyberScan ph/ion 510, Eutech Instruments, Vernon Hills, USA) and (ii) the increase of the cell number in Nutrient Broth by measuring the optical density of the cells at a wavelength of 600nm (OD 600 ) on a spectrophotometer with a diode field (LIBRA S6 Biochrom, Cambridge, UK). The cultivation was realised in Nutrient Broth without ornithine addition and also in Nutrient Broth with 0.2% (w/v) of ornithine. All of the results were submitted to statistical evaluation using the Unistat software (Student t-test, level of significance 5%). 3 Results and discussion Nutrient Broth was chosen for the cultivation of S. marcescens CCM 303. It is considered as suitable culture medium implementing growth and energetic requirements of undemanding microbial species. Further, biogenic amine production by various bacteria was proved in this medium before [14,15]. Before the determination of S. marcescens decarboxylase activity was accomplished, the effect of monosaccharide addition on ph of medium and bacteria growth was assessed both in medium without ornithine and with 0.2% (w/v) of ornithine. The addition of all monosaccharides caused in medium with initial ph 6 during cultivation ph decline ( ), especially in samples without ornithine (P<0.05). In the case of samples with ornithine the drop of ph was observed only in the presence of glucose ( ; P<0.05). The decrease of ph initiated by galactose and fructose was not significant ( and respectively; P 0.05). ph in medium with ornithine was higher than in medium without the amine (P<0.05). Culture medium ph was during bacterial growth significantly decreased (P<0.05) by glucose ( ) and fructose ( ) addition both in medium with and without ornithine at initial ph 7. There was no difference between 0.75 and 1.00% monosaccharide concentration (P 0.05). Galactose did not induced ph decline both in medium without ornithine ( ; P 0.05) and with the amino acid ( ; P 0.05). Samples with ornithine showed significantly greater drop of ph than samples without ornithine (P<0.05). Finally, during cultivation of S. marcescens CCM 303 in medium at ph 8, the glucose addition caused ph reduction in medium without ornithine ( ; P<0.05). In the case of fructose, ph fall was observed only up to 0.75% ( ; P<0.05). Addition of galactose did not evoke any depression of ph ( ; P 0.05). Decrease of ph was examined when glucose and fructose ( and respectively; P<0.05) was added to the medium with ornithine. Since glucose, fructose and galactose are ranked among simple fermentable saccharides, acids are produced by the action of bacteria thus lowering the ph. Addition of ornithine induced lesser decline of ph whereas S. marcescens metabolized ISBN:

3 monosaccharides and used ornithine as biogenic amine precursor (thus compensating ph drop) simultaneously. The presence of monosaccharides influenced also the growth of S. marcescens CCM 303 (data not shown). All quantities of monosaccharides ( % w/v) induced optical density rising (P<0.05) when cultivation at ph 6, 7 and 8 both in medium with and without ornithine. Particular monosaccharide concentration ( % w/v) did not affected the growth of the strain tested (P 0.05). The optical density showed higher values in the samples with ornithine than without the amino acid (P<0.05). The bacterial growth is influenced mainly by ph, temperature and duration of incubation [16]. Temperature and length of incubation (30 C and 20 h) were selected as common cultivation conditions for this type of bacteria. Similarly, Klebsiella pneumoniae from the same family Enterobacteriaceae produces the most biogenic amines at 30 and 37 C [17]. Putrescine production depending on the monosaccharide addition in the culture medium with initial ph 6 is depictured in Fig. 1. While glucose and galactose induced putrescine production increase only at the concentration 0.75% and 0.25% respectively (P<0.05), fructose raised putrescine formation at concentration % (P<0.05); other concentrations revealed to be inefficient (P 0.05). No unambiguous trend was observed between particular monosaccharide concentrations; values fluctuated in different ways. As the result of monosaccharide addition, putrescine production increased by mg/kg on average. exception (0.5%; P 0.05). Average decline was about mg/kg. Generally, differences between particular monosaccharide concentrations was detected (P<0.05). Putrescine formation in dependence on monosaccharide content in the culture medium with starting ph 8 is showed in Fig. 3. All of the monosaccharide concentration tested considerably lowered putrescine production (P<0.05). Rising quantity of glucose caused gradual reduction of putrescine amount (P<0.05). Fructose and galactose evoked successive inhibitory effect up to 0.75% and 0.5% respectively (P<0.05). However, even higher fructose and galactose concentration showed lower putrescine amount compared to zero-quantity of these monosaccharides (P<0.05). Putrescine creation lessened by mg/kg on average. Fig. 2. The effect of glucose, fructose and galactose medium with initial ph 7 Fig. 1. The effect of glucose, fructose and galactose medium with initial ph 6 The effect of glucose, fructose and galactose addition on the putrescine production at ph 7 is illustrated in Fig. 2. Adjunction of glucose provoked significant decrease in putrescine amount at all concentrations (P<0.05). Fructose and galactose reduced putrescine formation as well, with the only Fig. 3. The effect of glucose, fructose and galactose medium with initial ph 8 Putrescine production demonstrated that ph is the key factor influencing decarboxylase activity. This statement corresponds to previously published data [16]. The highest putrescine amount was detected in the culture medium with ph 7 ( mg/kg), followed by ph 8 ( mg/kg) and ph 6 ( mg/kg). Nevertheless, according to literature [16,18,19], decarboxylases show the maximum activity in acid environment (ph 4 5.5). This implies that biogenic amine formation is not ISBN:

4 only dependent on decarboxylase ph optimum, but also on bacteria growth activity [16]. Putrescine production was maximal at ph 7, which was optimal for S. marcescens growth. ph 6 seemed to be more suitable for decarboxylase function [16,18] resulting in putrescine production increase. Furthermore, putrescine acted as the protection from low ph which was not appropriate for S. marcescens growth. In the case of higher ph (less adequate for decarboxylases) monosaccharides were metabolised prior putrescine formation. Saccharides are important cell energy source as well as carbon sources for cell component synthesis. Different values which were obtained between particular monosaccharides are probably induced by distinct metabolic pathway of saccharides. Glucose-6-phosphate is important intermediate in metabolic pathways and it is essential in saccharide metabolism. The shortest metabolic pathway exhibits glucose while fructose and galactose degrade separately or through glucose in glycolysis [20]. Metabolic pathway affected unequal ph and optical density values too. Bacteria growth and decarboxylase activity is increased by 0.5 2% of saccharide [8]. Our results confirm these findings in the case of %. Further, it can be concluded that ornithine addition improved S. marcescens CCM 303 growth and putrescine formation which validated previously published study [4]. 4 Conclusion This work engaged in the determination of the effect of ph and selected monosaccharides on growth and decarboxylase activity (putrescine production) of S. marcescens CCM 303. It was stated that both ornithine and monosaccharide addition stimulated growth and also decarboxylase activity of S. marcescens CCM 303. ph 6 was found to be the most suitable for decarboxylase activity since monosaccharide adding intensified putrescine production. On the other hand, addition of monosaccharides at ph 7 and 8 caused inhibition of putrescine formation. The most extensive influence showed fructose, the least galactose. Acknowledgments: This study was supported by project of the internal grant of Tomas Bata University in Zlin No. IGA FT funded from the resources for specific university research. References: [1] HUI Y.H., Handbook of Food Science, Technology and Engineering, CRC Press, [2] HALÁSZ A., BARÁTH A., SIMON- SARKADI L., HOLZAPFEL W., Biogenic Amines and their Production by Microorganisms in Food, Trends in Food Science and Technology, Vol. 5, No. 2, 1994, pp [3] SILLA SANTOS M.H., Biogenic Amines: their Importance in Foods, International Journal of Food Microbiology, Vol. 29, No. 2 3, 1996, pp [4] BUŇKOVÁ L., BUŇKA F., KLČOVSKÁ P., MRKVIČKA V., DOLEŽALOVÁ M., KRÁČMAR S., Formation of Biogenic Amines by Gram-negative Bacteria Isolated from Poultry Skin, Food Chemistry, Vol. 121, No. 1, 2010, pp [5] JANDA J.M., ABBOT S.L., The Enterobacteria. ASP Press, [6] BOUCHEREAU A., AZIZ A., LARHER F., MARTIN-TANGUY, J., Polyamines and Environmental Challenges: Recent Development, Plant Science, Vol. 140, No. 2, 1999, pp [7] KALAČ P., KRAUSOVÁ P., A Review of Dietary Polyamines: Formation, Implications for Growth and Health and Occurrence in Foods, Food Chemistry, Vol. 90, No. 1 2, 2005, pp [8] SILLA SANTOS M.H., Toxic Nitrogen Compounds Produced during Processing: Biogenic Amines, Ethyl Carbamides, Nitrosamines, Aspen Publisher, [9] SHALABY A.R., Significance of Biogenic Amines to Food Safety and Human Health, Food Research International, Vol. 29, No. 7, 1996, pp [10] INNOCENTE N., BIASUTTI M., PADOVESE M., MORET S., Determination of Biogenic Amines in Cheese using HPLC Technique and Direct Derivatization of Acid Extract, Food Chemistry, Vol. 101, No. 3, 2007, pp [11] ÖNAL A., A Review: Current Analytical Methods for the Determination of Biogenic Amines in Foods, Food Chemistry, Vol. 103, No. 4, 2007, pp [12] NOUT M.J.R., Fermented Foods and Food Safety, Food Research International, Vol. 27, No. 3, 1994, pp [13] BUŇKOVÁ L., BUŇKA F., HLOBILOVÁ M., VAŇÁTKOVÁ Z., NOVÁKOVÁ D., ISBN:

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