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Polyamine determination in different strains of the yeast Debaryomyces hansenii by high pressure liquid chromatography
Food Chemistry 127 (2011) 1862–1865
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Polyamine determination in different strains of the yeast Debaryomyces hansenii by high pressure liquid chromatography Martha Reyes-Becerril a,b, Ma. Ángeles Esteban b, Dariel Tovar-Ramírez a, Felipe Ascencio-Valle a,⇑ a b
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain
a r t i c l e
i n f o
Article history: Received 5 October 2010 Received in revised form 5 January 2011 Accepted 1 February 2011 Available online 4 March 2011 Keywords: Polyamines Debaryomyces hansenii HPLC
a b s t r a c t Polyamines (putrescine, spermidine and spermine) participate in many biochemical processes that are indispensable for cell differentiation and proliferation. In the present study, polyamine types were determined in 13 Debaryomyces hansenii strains isolated from different sources by high pressure liquid chromatography. Very low putrescine levels were observed in the strains DhhBCS007, DhfBCS001 and DhfBCS002. Spermidine and spermine were the most prevalent amines detected in all the yeasts, ranging from 7.71 to 18.42 mg/kg of spermidine in the strain L2 and 9.39–18.96 mg/kg for spermine in strain DhhCBS004. D. hansenii strain L2 and CBS004 isolated from citrus fruit and marine water, respectively, were the main polyamine-secreting yeasts. The results will contribute to our ability to select the most appropriate strains for use, as dietary complement (probiotic) in the animal food industry, including aquaculture. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Polyamines or biologically active amines (putrescine, spermidine and spermine) are ubiquitous substances and present in every living cell (Bardocz et al., 1995). They have a low molecular weight, a simple chemical structure (aliphatic amines) and a high cationic charge in physiological conditions (Porat, Wender, Erez, & Kahana, 2005). Polyamines are essential for the maintenance of cell macromolecular biosynthesis, survival and growth and are involved in many cellular processes including the ion channelling function (Williams, 1997), nucleic acid packaging, DNA replication, transcription, translation and apoptosis (Ruan, Hill, Fatemie-Nainie, & Morris, 1994). Due to their important functions, polyamine levels are tightly regulated as low levels fail to support cell growth while excess appears to be toxic (Saito, Horie, Nose, Nakagomi, & Nakazawa, 1992). Different studies have brought to light the importance of polyamines from extracellular sources, including the diet and intestinal microorganisms (Bardocz, Brown, Grant, & Pusztai, 1990; Nishimura, Araki, Ohnishi, & Kozaki, 2001; Sarhan, Knodgen, & Seiler, 1989), intracellular polyamine concentrations effectively being regulated by their uptake by intestinal cells (Milovic, 2001), although some authors support the idea that the polyamines necessary for the growth of animal cells are synthesised in situ, that is by the cell itself (Kalac, Krızek, Pelikanova, Langova, & Veskrna, 2005). To date, despite the undoubted ⇑ Corresponding author. Tel.: +52 612 1238495; fax: +52 612 1253625. E-mail address: [email protected] (F. Ascencio-Valle). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.02.006
importance of dietary polyamines, very little information is available on the polyamine content and composition of even the most common foods (Kalac & Krausova, 2005). Debaryomyces spp. is a non-pathogenic and extremophilic yeast (Warren & Hazen, 1998) which occurs in many habitats with low water activity: e.g. hypersaline sites, such as seawater, from which it was initially isolated, soil and some foods (cheese, meat, wine, beer and fruits) (Barnett, Payne, & Yarrow, 2000). Debaryomyces hansenii has been investigated for its capacity to stimulate juvenile gilthead seabream (Sparus aurata) and leopard grouper (Mycteroperca rosacea) immune systems when administered in the diet (Reyes et al., 2008a,b). Its effect on the activity and expression of the main antioxidant enzymes in sea bass (Dicentrarchus labrax) larvae has also been evaluated. The results demonstrated that the group fed D. hansenii showed higher growth and lower glutathione peroxidase and superoxide dismutase activity and expression levels than fish fed a controlled diet, suggesting that the yeast is involved in superoxide anion retention in fish, which would be of importance to the host for increasing cell or tissue responsiveness to growth- and/or differentiation-enhancing factors (Tovar et al., 2010). These multiple beneficial effects could make yeasts and, more specifically, D. hansenii, promising probiotics. Taking into account all these considerations, the aim of the present work was to use high pressure liquid chromatography to determine the polyamine types and contents of thirteen strains of the yeast D. hansenii isolated from different sources. As the genus Debaryomyces has only been studied superficially, the present investigation will contribute to our knowledge of its potential
M. Reyes-Becerril et al. / Food Chemistry 127 (2011) 1862–1865
as a tool for tailoring its biotechnological production. The results will help in the selection of the most appropriate strains for use as dietary complement (probiotic) in the animal food industry, including aquaculture.
2. Materials and methods 2.1. Yeast strains and culture media The D. hansenii strains studied were from the yeast collection of the Centro de Investigaciones Biológicas del Noroeste (CIBNOR, Mexico), except the CBS 8339 strain which was isolated from the gut of rainbow trout reared in fresh water (Andlid, Vázquez-Juárez, & Gustafsson, 1995) (Table 1). Three strains were isolated from the surface of citrus fruit and cultured by cross-streaking on yeast extract peptone dextrose (YPD)-agar (containing 2.0% glucose, 2.0% peptone, 1.0% yeast extract and 1.5% agar prepared with distilled water and supplemented with 0.05% chloramphenicol) at 30 °C. The yeasts were removed from the lemon surface with a bacteriological loop and suspended in YPD-medium (100 ml in 1000 ml Erlenmeyer flasks) followed by a fresh incubation on a rotary shaker (100 rpm, 30 °C, 48 h) with constant aeration until the early stationary phase was reached. For the isolation of the nine marine yeasts, 2 ml of the seawater were suspended in 20 ml of YPD medium prepared with seawater and supplemented with 0.05% chloramphenicol, following the same procedure described above. Finally, yeast cells were removed from the growth medium by centrifugation (1000g, 5 min, 4 °C) and the pellet was recovered. The final cell concentration was adjusted to 1 107 colony forming units (CFU) (Sherman, 1991).
2.2. Sample preparation Fresh YDP broth (250 ml) was inoculated with 1 ml of D. hansenii followed by a fresh incubation on a rotary shaker with constant aeration (100 rpm, 30 °C, 24 h). One millilitre of medium was collected by centrifugation (2500g, 10 min, 4 °C), the pellet being recovered, suspended in 800 ll of 0.2 M HClO4 and vortexed. Samples were then sonicated on ice three times (10 s each time) at 100% power (12 watts/pin) (SonicMan™, Matrical, Inc., Spokane, WA, USA.) and the lysed yeasts were centrifuged (12,000g, 10 min, 4 °C). The supernatants were neutralised with a saturated solution of sodium carbonate (3 M) and dansylated for the reversed-phase high-performance liquid chromatography (HPLC) assay.
Table 1 Collection of yeast D. hansenii using in the present study. Strain
Description
CBS 8339 L1 L2 L3 Dhf BCS 001 Dhf BCS 002 DhhBCS 001 DhhBCS 002 DhhBCS 003 DhhBCS 004 DhhBCS 005 DhhBCS 006 DhhBCS 007
Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces
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2.3. Reagents and standards Putrescine dihydrochloride (put), spermine tetrahydrochloride (spm), spermidine trihydrochloride (spd) and 1,3-diaminopropane dihydrochloride (diam) as internal standard (ALDRICH D2, 380-7, St. Louis, United States) were dissolved separately in HPLC grade water. The final concentration of free base for each amine was 8 mg ml1. Biogenic amine standards were purchased from Sigma Aldrich (P 7505, S 2876, S 0381). Dansyl chloride (5-dimethylaminonaphthalene-1-sulphonylchloride, >98%) and trichloroacetic acid (>99%) were obtained from Fluka Biochemika (Buchs, Switzerland), while acetonitrile (ACN) of HPLC grade and perchloric acid (69–72%) were obtained from J.T. Baker (Phillipsburg, USA). Acetic acid, anhydrous sodium carbonate, CHROMASOLVÒ for HPLC grade water and Toluene grade HPLC (99.8%) were obtained from SIGMA (St. Louis, United States). 2.4. Chromatographic system and equipment High-performance liquid chromatography analyses were performed using a Waters model 2695. Fluorescence excitation was at 333 nm, and emission at 390 nm. The LC-18-DB columns (150 5.0 mm, particle diameter 5 lm) were purchased from SUPELCOSIL (SIGMA–ALDRICH). Separation after dansyl chloride derivatization was carried out by gradient elution with H2O/ACN (time 0–24 min: H2O 40–0%, ACN 60–100%) at a flow rate of 1 ml min1. 2.5. Polyamine determination The polyamine content of the yeast was examined by HPLC, according to Malle, Valle, and Bouquelet (1996) with some modifications. Briefly, after sample preparation (see Section 2.2), 100 ll the supernatant was removed and the cells were resuspended in 100 ll diam solution, 200 ll saturated NaCO3 was added, followed by 400 ll of 0.027 M dansyl chloride solution. Mixtures were vortexed and incubated in a water bath (60 °C, 20 min). Aliquots of 100 ll of 0.8 M proline solution were added and the mixture was vortexed and allowed to stand at 25 °C for 30 min in the dark. Toluene (500 ll) was added to each sample and the mixtures were mixed and incubated at 25 °C for another 10 min. The organic phase was recovered and evaporated under a stream of nitrogen. The pellets were resuspended in 300 ll acetonitrile, filtered through a 0.2 lm in an AcrodiscÒ (VWR) and finally 20 ll aliquots were injected into the HPLC. The resultant dansylated polyamines were detected by means of a fluorescence detector (420-AC; Waters Chromatography, Milford, MA). The absorbance of derivative polyamines was measured at 254 nm. Prior to sample analysis, pattern curves were prepared with putrescine chlorohydrate (99% purity), spermidine trichlordidrate (98% purity) and tetrahydrochlorohydrate of spermine (95% purity). The results are presented in mg kg1.
Origin hansenii hansenii hansenii hansenii hansenii hansenii hansenii hansenii hansenii hansenii hansenii hansenii hansenii
var. var. var. var. var. var. var. var. var.
fabryi fabryi hansenii hansenii hansenii hansenii hansenii hansenii hansenii
(Y-GRT) (Y-CF) (Y-CF) (Y-CF) (Y-MW) (Y-MW) (Y-MW) (Y-MW) (Y-MW) (Y-MW) (Y-MW) (Y-MW) (Y-MW)
Y-GRT, Yeast isolated from gut of rainbow trout; Y-CF, Yeast isolated from citrus fruit; Y-MW, Yeast isolated from marine water.
2.6. Polyamine quantification The correlation coefficient (r) of peak areas for each compound was calculated against amine standard concentrations after injecting six replicates of each standard solution of amine. Linear leastsquares regression was used to calculate the slopes, intercepts, and correlation coefficients. The concentration of biogenic amine in each sample (cx) was calculated as follows:
cx ¼ RFx ðcIS AxÞ=AIS where: RFx = response factor of the amine (RFx = AIS/Ax cx/cIS), AIS = peak area of the internal standard, Ax = peak area of biogenic amine, cIS = concentration of the internal standard.
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1,3-Diaminopropane dihydrochloride was used as internal standard (IS) because the whole procedure involves multiple steps, the data being used for correcting the recovery (R) measurements. The recovery was calculated as:
%R ¼ ½ðCF CUÞ=CA 100 where: CF = concentration of the spiked sample, CU = concentration of the original sample, CA = concentration of the analyte added. 2.7. Statistical analysis Data are presented as mean ± standard deviation. An analysis of variance (ANOVA) was carried out to verify statistically significant differences between the means. In addition, Tukey’s HSD test (honestly significant differences) for multiple comparisons was used and the differences between the means were considered statistically significant for P-values of 60.05. All statistical analyses were performed using the SPSS statistic programme v.17.0 software (SPSS Inc., 233 South Wacker Drive, 11th Floor, Chicago, IL).
Table 3 Response factors (RF) of amines. Strain
RFa DhCBS8339 L1 L2 L3 DhfBCS001 DhfBCS002 DhhBCS001 DhhBCS002 DhhBCS003 DhhBCS004 DhhBCS005 DhhBCS006 DhhBCS007
Polyamine Putrescine
Spermidine
Spermine
2.776 ± 0.069 2.508 ± 0.056 0.886 ± 0.074 0.570 ± 0.023 0.409 ± 0.021 0.599 ± 0.016 2.774 ± 0.012 1.338 ± 0.022 0.992 ± 0.046 1.050 ± 0.044 1.041 ± 0.056 0.958 ± 0.025 0.647 ± 0.012
0.245 ± 0.013 0.384 ± 0.016 0.150 ± 0.009 0.149 ± 0.011 0.391 ± 0.010 0.264 ± 0.011 0.739 ± 0.006 0.778 ± 0.023 0.834 ± 0.064 0.822 ± 0.022 0.847 ± 0.013 0.837 ± 0.012 3.395 ± 0.078
0.834 ± 0.056 0.185 ± 0.010 0.155 ± 0.009 0.104 ± 0.011 1.486 ± 0.052 1.674 ± 0.025 4.789 ± 0.091 5.767 ± 0.099 3.723 ± 0.079 0.670 ± 0.020 2.270 ± 0.077 2.310 ± 0.056 0.986 ± 0.033
a RF = [(I.S. area/standard amine area) (standard amine concentration)/I.S. concentration]. Here I.S. = Internal Standard. Mean ± standard deviation, for three replicates of the same sample, n = 3.
3. Results and discussion Polyamines are ancient small polycation metabolites that are essential for cell growth and proliferation and are found in most, if not all, bacterial, fungal, plant and animal cells (Cohen, 1998). In the present study thirteen strains of the yeast D. hansenii were evaluated for their capacity to produce polyamines (putrescine, spermine and spermidine) by HPLC (Table 1). According to present day taxonomy, two varieties of D. hansenii can be distinguished (D. hansenii var. fabryi and D. hansenii var. hansenii), with different properties: e.g. var. hansenii can only grow at temperatures below 35 °C while var. fabryi can grow up to 39 °C (Breuer & Harms, 2006). In the present research, two strains of the var. fabryi and seven of D. hansenii were studied. The 13 yeasts were cultured at 30 °C for 24 h in YPD-medium. To calculate the recovery, the concentrations determined before and after the standard addition were compared. The recoveries of each biogenic amine spiked at four levels (0.5, 1.0, 1.5 and 2.0 lg/ml) are presented in Table 2. The best recovery was for the concentration 1.0 lg/ml in the range of 75% for putrescine to 85% for spermidine. At higher concentrations (2.0 lg/ml), amine recovery was lower. Recoveries exceeding 100% are normal, while recoveries exceeding 105% suggest equipment problems (column and detector) (Baston, Stroia, Moise, & Barna, 2008). The response factor (RF) value was used to calculate the polyamine concentration and the obtained data are presented in Table 3. Polyamine concentrations in the different strains of D. hansenii are shown in Fig. 1. The three polyamines (putrescine, spermidine and spermine) were detected in the 13 yeasts, but the amounts retrieved in D. hansenii L2 and DhhBCS004 were significantly higher than those present in the other strains. Spermine and spermidine
Table 2 Recovery (%) of amines during extraction and purification by a standard addition technique. Recovery (%) Concentration lg/ml Polyamines Putrescine Spermidine Spermine
0.5 103.4 ± 2.013 109.0 ± 4.503 105.2 ± 2.081
1.0 75.0 ± 3.605 85.0 ± 2.003 80.2 ± 4.582
1.5 87.33 ± 3.750 52.66 ± 3.288 87.33 ± 3.501
2.0 42.75 ± 3.573 68.50 ± 2.384 47.75 ± 2.179
Yeast sample spiked with the mixture of polyamines standards at 0.5, 1.0, 1.5 and 2 lg/ml (measure was done three times).
were the main polyamines produced in all the studied strains while the levels of putrescine were always low. These results contrast with those obtained for several groups of bacteria, mainly Enterobacteriaceae and Clostridium spp., which had a high putrescine content (Shalaby, 1996). Tovar et al. (2002) demonstrated that the production of the three polyamines by D. hansenii HFI (isolated from rainbow trout) was higher than that produced by Saccharomyces cerevisiae X2180 (reference strain). In other words although bacteria produce more polyamines than yeast, the genus Debaryomyces produced more polyamines than other yeast genera, such as Saccharomyces. In Tovar’s study spermine and spermidine were extensively secreted from the cells (6.38 and 17.33 nM ml1, respectively), whereas putrescine remained at a relatively low level, especially in S. cerevisiae. In the same work, the role of polyamine in gut maturation in sea bass larvae was demonstrated by the incorporation of D. hansenii HFI in the diet (Tovar et al., 2002); similarly, polyamines are vital for the functioning and renewal of the gut epithelium in rat (Milovic, 2001). The present results emphasise that the yeast D. hansenii is an important producer of spermidine and spermine. Spermidine reached its highest average level in L2 (up to 18.42 mg/kg). In plant foods spermidine levels are commonly higher than those of spermine (Kalac and Krausova, 2005). Eisenberg et al. (2009) proposed that spermidine promotes longevity by upregulating the lysosomal/vacuolar degradation pathway (referred to as autophagy), leading to an enhanced resistance to oxidative stress and lower levels of cell death in yeast. Furthermore, it has been demonstrated that spermidine can bind (approximately 60%) with RNA to modulate mRNA translation in lymphocytes (Igarashi and Kashiwagi, 2010). In our study, spermine concentrations reached up to 18.96 mg/kg in DhhBCS004, similar to that found for spermidine in the strain L2. Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesised from spermidine. The polyamines spermidine and spermine from extracellular sources are very important, since polyamines are partly metabolized during the absorption process and putrescine is more readily metabolized than spermidine or spermine (Bardocz et al., 1995). One hour after putrescine administration, 80% of putrescine was converted into non-polyamine metabolites, especially amino acids, whereas 70–80% of spermidine and spermine remained in their original forms after spermidine and spermine administration (Bardocz et al., 1995).
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Fig. 1. Polyamine content in 13 D. hansenii strains. h Putrescine; j Spermidine; M Spermine. Data represents mean ± standard deviation for three replicates of the same sample (n = 3). Different superscripts in the same column indicate statistically significant differences (p < 0.05).
From this study it can be concluded that D. hansenii strains DhhBCS004 and L2 (isolated from marine water and citrus fruit, respectively) are especially rich in spermidine and spermine. Since polyamines play a crucial role in cell proliferation and differentiation, these D. hansenii strains could be included among potential biological control agents when administered in the diet as additives or probiotics. Further extensive trials with different target animals and a commercial cost-benefit analysis are necessary to establish their suitability for use as a candidate probiotic in disease prevention and/or growth stimulator of farm animals. Acknowledgements We thank Orlando Lugo for technical support in the HPLC assays. The first author received postdoctoral scholarships from the Mexican Consejo Nacional de Ciencia y Tecnología (CONACYT Grant 164653). This work was funded partly by grants from Ministerio de Ciencia e Innovación (AGL2008-05119-C02-01, Spain) and Fundación Séneca of Murcia, Spain (04538/GERM/06). References Andlid, T., Vázquez-Juárez, R., & Gustafsson, L. (1995). Yeast colonising the intestine of rainbowtrout (Salmo gairdneri) and turbot (Scophtalmus maximus). Microbiology Ecology, 30, 321–324. Bardocz, S., Brown, D. S., Grant, G., & Pusztai, A. (1990). Luminal and basolateral polyamine uptake by rat small intestine stimulated to grow by Phaseolus vulgaris lectin phytohaemagglutinin in vivo. Biochemical Biophysica Acta, 1034, 46–52. Bardocz, S., Duguid, Z. J., Brown, D. S., Grant, G., Pusztai, A., White, A., et al. (1995). The importance of dietary polyamines in cell regeneration and growth. British Journal of Nutrition, 73, 819–828. Barnett, J. A., Payne, R. W., & Yarrow, D. (2000). In J. A. Barnett, R. W. Payne, & D. Yarrow (Eds.), Yeasts: characteristics, and identification (3rd ed.). Cambridge: Cambridge University Press. Baston, O., Stroia, A. L., Moise, D., & Barna, O. (2008). Validation study of a HPLC method able to measure biogenic amines in chicken meat. Food Technology, 31, 44–50. Breuer, U., & Harms, H. (2006). Debaryomyces hansenii-an extremophilic yeast with biotechnological potential. Yeast, 23, 415–437. Cohen, S. (1998). A guide to the polyamines. Oxford: Oxford University Press. pp. 1–543. Eisenberg, T., Knauer, H., Schauer, A., Fussi, H., Büttner, S., Ruckenstuhl, C., et al. (2009). Induction of autophagy by spermidine promotes longevity. Nature Cell Biology, 11, 1305–1314.
Igarashi, K., & Kashiwagi, K. (2010). Modulation of cellular function by polyamines. International Journal of Biochemistry & Cell Biology, 42, 39–51. Kalac, P., & Krausova, P. (2005). A review of dietary polyamines: Formation, implications for growth and health and occurrence in foods. Food Chemistry, 90, 219–230. Kalac, P., Krızek, M., Pelikanova, T., Langova, M., & Veskrna, O. (2005). Contents of polyamines in selected foods. Food Chemistry, 90, 561–564. Malle, P., Valle, M., & Bouquelet, S. (1996). Assay of biogenic amines involved in fish decomposition. Journal AOAC International, 79, 43–49. Milovic, V. (2001). Polyamines in the gut lumen: Bioavailability and biodistribution. European Journal of Gastroenterology and Hepatology, 13, 1021–1025. Nishimura, K., Araki, N., Ohnishi, Y., & Kozaki, S. (2001). Effects of dietary polyamine deficiency on Trypanosoma gambiense infection in rats. Experimental Parasitology, 97, 95–101. Porat, Z., Wender, N., Erez, O., & Kahana, C. (2005). Mechanism of polyamine tolerance in yeast: novel regulators and insights. Cellular Molecular Life Science, 62, 3106–3116. Reyes, B. M., Salinas, I., Cuesta, A., Meseguer, J., Tovar, R. D., Ascencio, V. F., et al. (2008a). Oral delivery of live yeast Debaryomyces hansenii modulates the main innate immune parameters and the expression of immune-relevant genes in the gilthead seabream (Sparus aurata L.). Fish Shellfish Immunology, 25, 433–438. Reyes, B. M., Tovar, R. D., Ascencio, V. F., Civera, C. R., Gracia, L. V., & Barbosa, S. V. (2008b). Effects of dietary live yeast Debaryomyces hansenii on the immune and antioxidant system in juvenile leopard grouper Mycteroperca rosacea exposed to stress. Aquaculture, 280, 39–44. Ruan, H., Hill, J. R., Fatemie-Nainie, S., & Morris, D. R. (1994). Cell-specific translational regulation of S-adenosylmethionine decarboxylase mRNA. Influence of the structure of the 5´transcript leader on regulation by the upstream open reading frame. Journal of Biological Chemistry, 269, 17905–17910. Saito, K., Horie, M., Nose, N., Nakagomi, K., & Nakazawa, H. (1992). Determination of polyamines in foods by liquid chromatography with on-column fluorescence derivatization. Analytical Sciences, 8, 675–680. Sarhan, S., Knodgen, B., & Seiler, N. (1989). The gastrointestinal tract as polyamine source for tumour growth. Anticancer Research, 9, 215–224. Shalaby, A. R. (1996). Significance of biogenic amines to food safety and human health. Food Research International, 29, 675–690. Sherman, F. (1991). Getting started with yeast. In Ch. Guthrie & G. R. Fink (Eds.). Method in enzymology. Guide to yeast genetics and molecular biology (Vol. 194, pp. 3–21). New York: Academic Press. Tovar, R. D., Mazurais, D., Gatesoupe, J. F., Quazuguel, P., Cahu, C. L., & ZamboninoInfante, J. L. (2010). Dietary probiotic live yeast modulates antioxidant enzyme activities and gene expression of sea bass (Dicentrarchus labrax) larvae. Aquaculture, 300, 142–147. Tovar, D., Zambonino, J., Cahu, C., Gatesoupe, F. J., Vázquez, J. R., & Lésel, R. (2002). Effect of live yeast incorporation in compound diet on digestive enzyme activity in sea bass (Dicentrarchus labrax) larvae. Aquaculture, 204, 113–123. Warren, N. G., & Hazen, K. C. (1998). Candida, cryptococcus, and other yeasts of medical importance. In P. R. Murray (Ed.), Manual of clinical microbiology (7th ed., pp. 1184–1199). Washington D.C.: ASM Press. Williams, K. (1997). Interactions of polyamines with ion channels. Biochemical Journal, 325, 289–297.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Polyamine determination in different strains of the yeast Debaryomyces hansenii by high pressure liquid chromatography Martha Reyes-Becerril a,b, Ma. Ángeles Esteban b, Dariel Tovar-Ramírez a, Felipe Ascencio-Valle a,⇑ a b
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain
a r t i c l e
i n f o
Article history: Received 5 October 2010 Received in revised form 5 January 2011 Accepted 1 February 2011 Available online 4 March 2011 Keywords: Polyamines Debaryomyces hansenii HPLC
a b s t r a c t Polyamines (putrescine, spermidine and spermine) participate in many biochemical processes that are indispensable for cell differentiation and proliferation. In the present study, polyamine types were determined in 13 Debaryomyces hansenii strains isolated from different sources by high pressure liquid chromatography. Very low putrescine levels were observed in the strains DhhBCS007, DhfBCS001 and DhfBCS002. Spermidine and spermine were the most prevalent amines detected in all the yeasts, ranging from 7.71 to 18.42 mg/kg of spermidine in the strain L2 and 9.39–18.96 mg/kg for spermine in strain DhhCBS004. D. hansenii strain L2 and CBS004 isolated from citrus fruit and marine water, respectively, were the main polyamine-secreting yeasts. The results will contribute to our ability to select the most appropriate strains for use, as dietary complement (probiotic) in the animal food industry, including aquaculture. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Polyamines or biologically active amines (putrescine, spermidine and spermine) are ubiquitous substances and present in every living cell (Bardocz et al., 1995). They have a low molecular weight, a simple chemical structure (aliphatic amines) and a high cationic charge in physiological conditions (Porat, Wender, Erez, & Kahana, 2005). Polyamines are essential for the maintenance of cell macromolecular biosynthesis, survival and growth and are involved in many cellular processes including the ion channelling function (Williams, 1997), nucleic acid packaging, DNA replication, transcription, translation and apoptosis (Ruan, Hill, Fatemie-Nainie, & Morris, 1994). Due to their important functions, polyamine levels are tightly regulated as low levels fail to support cell growth while excess appears to be toxic (Saito, Horie, Nose, Nakagomi, & Nakazawa, 1992). Different studies have brought to light the importance of polyamines from extracellular sources, including the diet and intestinal microorganisms (Bardocz, Brown, Grant, & Pusztai, 1990; Nishimura, Araki, Ohnishi, & Kozaki, 2001; Sarhan, Knodgen, & Seiler, 1989), intracellular polyamine concentrations effectively being regulated by their uptake by intestinal cells (Milovic, 2001), although some authors support the idea that the polyamines necessary for the growth of animal cells are synthesised in situ, that is by the cell itself (Kalac, Krızek, Pelikanova, Langova, & Veskrna, 2005). To date, despite the undoubted ⇑ Corresponding author. Tel.: +52 612 1238495; fax: +52 612 1253625. E-mail address: [email protected] (F. Ascencio-Valle). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.02.006
importance of dietary polyamines, very little information is available on the polyamine content and composition of even the most common foods (Kalac & Krausova, 2005). Debaryomyces spp. is a non-pathogenic and extremophilic yeast (Warren & Hazen, 1998) which occurs in many habitats with low water activity: e.g. hypersaline sites, such as seawater, from which it was initially isolated, soil and some foods (cheese, meat, wine, beer and fruits) (Barnett, Payne, & Yarrow, 2000). Debaryomyces hansenii has been investigated for its capacity to stimulate juvenile gilthead seabream (Sparus aurata) and leopard grouper (Mycteroperca rosacea) immune systems when administered in the diet (Reyes et al., 2008a,b). Its effect on the activity and expression of the main antioxidant enzymes in sea bass (Dicentrarchus labrax) larvae has also been evaluated. The results demonstrated that the group fed D. hansenii showed higher growth and lower glutathione peroxidase and superoxide dismutase activity and expression levels than fish fed a controlled diet, suggesting that the yeast is involved in superoxide anion retention in fish, which would be of importance to the host for increasing cell or tissue responsiveness to growth- and/or differentiation-enhancing factors (Tovar et al., 2010). These multiple beneficial effects could make yeasts and, more specifically, D. hansenii, promising probiotics. Taking into account all these considerations, the aim of the present work was to use high pressure liquid chromatography to determine the polyamine types and contents of thirteen strains of the yeast D. hansenii isolated from different sources. As the genus Debaryomyces has only been studied superficially, the present investigation will contribute to our knowledge of its potential
M. Reyes-Becerril et al. / Food Chemistry 127 (2011) 1862–1865
as a tool for tailoring its biotechnological production. The results will help in the selection of the most appropriate strains for use as dietary complement (probiotic) in the animal food industry, including aquaculture.
2. Materials and methods 2.1. Yeast strains and culture media The D. hansenii strains studied were from the yeast collection of the Centro de Investigaciones Biológicas del Noroeste (CIBNOR, Mexico), except the CBS 8339 strain which was isolated from the gut of rainbow trout reared in fresh water (Andlid, Vázquez-Juárez, & Gustafsson, 1995) (Table 1). Three strains were isolated from the surface of citrus fruit and cultured by cross-streaking on yeast extract peptone dextrose (YPD)-agar (containing 2.0% glucose, 2.0% peptone, 1.0% yeast extract and 1.5% agar prepared with distilled water and supplemented with 0.05% chloramphenicol) at 30 °C. The yeasts were removed from the lemon surface with a bacteriological loop and suspended in YPD-medium (100 ml in 1000 ml Erlenmeyer flasks) followed by a fresh incubation on a rotary shaker (100 rpm, 30 °C, 48 h) with constant aeration until the early stationary phase was reached. For the isolation of the nine marine yeasts, 2 ml of the seawater were suspended in 20 ml of YPD medium prepared with seawater and supplemented with 0.05% chloramphenicol, following the same procedure described above. Finally, yeast cells were removed from the growth medium by centrifugation (1000g, 5 min, 4 °C) and the pellet was recovered. The final cell concentration was adjusted to 1 107 colony forming units (CFU) (Sherman, 1991).
2.2. Sample preparation Fresh YDP broth (250 ml) was inoculated with 1 ml of D. hansenii followed by a fresh incubation on a rotary shaker with constant aeration (100 rpm, 30 °C, 24 h). One millilitre of medium was collected by centrifugation (2500g, 10 min, 4 °C), the pellet being recovered, suspended in 800 ll of 0.2 M HClO4 and vortexed. Samples were then sonicated on ice three times (10 s each time) at 100% power (12 watts/pin) (SonicMan™, Matrical, Inc., Spokane, WA, USA.) and the lysed yeasts were centrifuged (12,000g, 10 min, 4 °C). The supernatants were neutralised with a saturated solution of sodium carbonate (3 M) and dansylated for the reversed-phase high-performance liquid chromatography (HPLC) assay.
Table 1 Collection of yeast D. hansenii using in the present study. Strain
Description
CBS 8339 L1 L2 L3 Dhf BCS 001 Dhf BCS 002 DhhBCS 001 DhhBCS 002 DhhBCS 003 DhhBCS 004 DhhBCS 005 DhhBCS 006 DhhBCS 007
Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces Debaryomyces
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2.3. Reagents and standards Putrescine dihydrochloride (put), spermine tetrahydrochloride (spm), spermidine trihydrochloride (spd) and 1,3-diaminopropane dihydrochloride (diam) as internal standard (ALDRICH D2, 380-7, St. Louis, United States) were dissolved separately in HPLC grade water. The final concentration of free base for each amine was 8 mg ml1. Biogenic amine standards were purchased from Sigma Aldrich (P 7505, S 2876, S 0381). Dansyl chloride (5-dimethylaminonaphthalene-1-sulphonylchloride, >98%) and trichloroacetic acid (>99%) were obtained from Fluka Biochemika (Buchs, Switzerland), while acetonitrile (ACN) of HPLC grade and perchloric acid (69–72%) were obtained from J.T. Baker (Phillipsburg, USA). Acetic acid, anhydrous sodium carbonate, CHROMASOLVÒ for HPLC grade water and Toluene grade HPLC (99.8%) were obtained from SIGMA (St. Louis, United States). 2.4. Chromatographic system and equipment High-performance liquid chromatography analyses were performed using a Waters model 2695. Fluorescence excitation was at 333 nm, and emission at 390 nm. The LC-18-DB columns (150 5.0 mm, particle diameter 5 lm) were purchased from SUPELCOSIL (SIGMA–ALDRICH). Separation after dansyl chloride derivatization was carried out by gradient elution with H2O/ACN (time 0–24 min: H2O 40–0%, ACN 60–100%) at a flow rate of 1 ml min1. 2.5. Polyamine determination The polyamine content of the yeast was examined by HPLC, according to Malle, Valle, and Bouquelet (1996) with some modifications. Briefly, after sample preparation (see Section 2.2), 100 ll the supernatant was removed and the cells were resuspended in 100 ll diam solution, 200 ll saturated NaCO3 was added, followed by 400 ll of 0.027 M dansyl chloride solution. Mixtures were vortexed and incubated in a water bath (60 °C, 20 min). Aliquots of 100 ll of 0.8 M proline solution were added and the mixture was vortexed and allowed to stand at 25 °C for 30 min in the dark. Toluene (500 ll) was added to each sample and the mixtures were mixed and incubated at 25 °C for another 10 min. The organic phase was recovered and evaporated under a stream of nitrogen. The pellets were resuspended in 300 ll acetonitrile, filtered through a 0.2 lm in an AcrodiscÒ (VWR) and finally 20 ll aliquots were injected into the HPLC. The resultant dansylated polyamines were detected by means of a fluorescence detector (420-AC; Waters Chromatography, Milford, MA). The absorbance of derivative polyamines was measured at 254 nm. Prior to sample analysis, pattern curves were prepared with putrescine chlorohydrate (99% purity), spermidine trichlordidrate (98% purity) and tetrahydrochlorohydrate of spermine (95% purity). The results are presented in mg kg1.
Origin hansenii hansenii hansenii hansenii hansenii hansenii hansenii hansenii hansenii hansenii hansenii hansenii hansenii
var. var. var. var. var. var. var. var. var.
fabryi fabryi hansenii hansenii hansenii hansenii hansenii hansenii hansenii
(Y-GRT) (Y-CF) (Y-CF) (Y-CF) (Y-MW) (Y-MW) (Y-MW) (Y-MW) (Y-MW) (Y-MW) (Y-MW) (Y-MW) (Y-MW)
Y-GRT, Yeast isolated from gut of rainbow trout; Y-CF, Yeast isolated from citrus fruit; Y-MW, Yeast isolated from marine water.
2.6. Polyamine quantification The correlation coefficient (r) of peak areas for each compound was calculated against amine standard concentrations after injecting six replicates of each standard solution of amine. Linear leastsquares regression was used to calculate the slopes, intercepts, and correlation coefficients. The concentration of biogenic amine in each sample (cx) was calculated as follows:
cx ¼ RFx ðcIS AxÞ=AIS where: RFx = response factor of the amine (RFx = AIS/Ax cx/cIS), AIS = peak area of the internal standard, Ax = peak area of biogenic amine, cIS = concentration of the internal standard.
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1,3-Diaminopropane dihydrochloride was used as internal standard (IS) because the whole procedure involves multiple steps, the data being used for correcting the recovery (R) measurements. The recovery was calculated as:
%R ¼ ½ðCF CUÞ=CA 100 where: CF = concentration of the spiked sample, CU = concentration of the original sample, CA = concentration of the analyte added. 2.7. Statistical analysis Data are presented as mean ± standard deviation. An analysis of variance (ANOVA) was carried out to verify statistically significant differences between the means. In addition, Tukey’s HSD test (honestly significant differences) for multiple comparisons was used and the differences between the means were considered statistically significant for P-values of 60.05. All statistical analyses were performed using the SPSS statistic programme v.17.0 software (SPSS Inc., 233 South Wacker Drive, 11th Floor, Chicago, IL).
Table 3 Response factors (RF) of amines. Strain
RFa DhCBS8339 L1 L2 L3 DhfBCS001 DhfBCS002 DhhBCS001 DhhBCS002 DhhBCS003 DhhBCS004 DhhBCS005 DhhBCS006 DhhBCS007
Polyamine Putrescine
Spermidine
Spermine
2.776 ± 0.069 2.508 ± 0.056 0.886 ± 0.074 0.570 ± 0.023 0.409 ± 0.021 0.599 ± 0.016 2.774 ± 0.012 1.338 ± 0.022 0.992 ± 0.046 1.050 ± 0.044 1.041 ± 0.056 0.958 ± 0.025 0.647 ± 0.012
0.245 ± 0.013 0.384 ± 0.016 0.150 ± 0.009 0.149 ± 0.011 0.391 ± 0.010 0.264 ± 0.011 0.739 ± 0.006 0.778 ± 0.023 0.834 ± 0.064 0.822 ± 0.022 0.847 ± 0.013 0.837 ± 0.012 3.395 ± 0.078
0.834 ± 0.056 0.185 ± 0.010 0.155 ± 0.009 0.104 ± 0.011 1.486 ± 0.052 1.674 ± 0.025 4.789 ± 0.091 5.767 ± 0.099 3.723 ± 0.079 0.670 ± 0.020 2.270 ± 0.077 2.310 ± 0.056 0.986 ± 0.033
a RF = [(I.S. area/standard amine area) (standard amine concentration)/I.S. concentration]. Here I.S. = Internal Standard. Mean ± standard deviation, for three replicates of the same sample, n = 3.
3. Results and discussion Polyamines are ancient small polycation metabolites that are essential for cell growth and proliferation and are found in most, if not all, bacterial, fungal, plant and animal cells (Cohen, 1998). In the present study thirteen strains of the yeast D. hansenii were evaluated for their capacity to produce polyamines (putrescine, spermine and spermidine) by HPLC (Table 1). According to present day taxonomy, two varieties of D. hansenii can be distinguished (D. hansenii var. fabryi and D. hansenii var. hansenii), with different properties: e.g. var. hansenii can only grow at temperatures below 35 °C while var. fabryi can grow up to 39 °C (Breuer & Harms, 2006). In the present research, two strains of the var. fabryi and seven of D. hansenii were studied. The 13 yeasts were cultured at 30 °C for 24 h in YPD-medium. To calculate the recovery, the concentrations determined before and after the standard addition were compared. The recoveries of each biogenic amine spiked at four levels (0.5, 1.0, 1.5 and 2.0 lg/ml) are presented in Table 2. The best recovery was for the concentration 1.0 lg/ml in the range of 75% for putrescine to 85% for spermidine. At higher concentrations (2.0 lg/ml), amine recovery was lower. Recoveries exceeding 100% are normal, while recoveries exceeding 105% suggest equipment problems (column and detector) (Baston, Stroia, Moise, & Barna, 2008). The response factor (RF) value was used to calculate the polyamine concentration and the obtained data are presented in Table 3. Polyamine concentrations in the different strains of D. hansenii are shown in Fig. 1. The three polyamines (putrescine, spermidine and spermine) were detected in the 13 yeasts, but the amounts retrieved in D. hansenii L2 and DhhBCS004 were significantly higher than those present in the other strains. Spermine and spermidine
Table 2 Recovery (%) of amines during extraction and purification by a standard addition technique. Recovery (%) Concentration lg/ml Polyamines Putrescine Spermidine Spermine
0.5 103.4 ± 2.013 109.0 ± 4.503 105.2 ± 2.081
1.0 75.0 ± 3.605 85.0 ± 2.003 80.2 ± 4.582
1.5 87.33 ± 3.750 52.66 ± 3.288 87.33 ± 3.501
2.0 42.75 ± 3.573 68.50 ± 2.384 47.75 ± 2.179
Yeast sample spiked with the mixture of polyamines standards at 0.5, 1.0, 1.5 and 2 lg/ml (measure was done three times).
were the main polyamines produced in all the studied strains while the levels of putrescine were always low. These results contrast with those obtained for several groups of bacteria, mainly Enterobacteriaceae and Clostridium spp., which had a high putrescine content (Shalaby, 1996). Tovar et al. (2002) demonstrated that the production of the three polyamines by D. hansenii HFI (isolated from rainbow trout) was higher than that produced by Saccharomyces cerevisiae X2180 (reference strain). In other words although bacteria produce more polyamines than yeast, the genus Debaryomyces produced more polyamines than other yeast genera, such as Saccharomyces. In Tovar’s study spermine and spermidine were extensively secreted from the cells (6.38 and 17.33 nM ml1, respectively), whereas putrescine remained at a relatively low level, especially in S. cerevisiae. In the same work, the role of polyamine in gut maturation in sea bass larvae was demonstrated by the incorporation of D. hansenii HFI in the diet (Tovar et al., 2002); similarly, polyamines are vital for the functioning and renewal of the gut epithelium in rat (Milovic, 2001). The present results emphasise that the yeast D. hansenii is an important producer of spermidine and spermine. Spermidine reached its highest average level in L2 (up to 18.42 mg/kg). In plant foods spermidine levels are commonly higher than those of spermine (Kalac and Krausova, 2005). Eisenberg et al. (2009) proposed that spermidine promotes longevity by upregulating the lysosomal/vacuolar degradation pathway (referred to as autophagy), leading to an enhanced resistance to oxidative stress and lower levels of cell death in yeast. Furthermore, it has been demonstrated that spermidine can bind (approximately 60%) with RNA to modulate mRNA translation in lymphocytes (Igarashi and Kashiwagi, 2010). In our study, spermine concentrations reached up to 18.96 mg/kg in DhhBCS004, similar to that found for spermidine in the strain L2. Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesised from spermidine. The polyamines spermidine and spermine from extracellular sources are very important, since polyamines are partly metabolized during the absorption process and putrescine is more readily metabolized than spermidine or spermine (Bardocz et al., 1995). One hour after putrescine administration, 80% of putrescine was converted into non-polyamine metabolites, especially amino acids, whereas 70–80% of spermidine and spermine remained in their original forms after spermidine and spermine administration (Bardocz et al., 1995).
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Fig. 1. Polyamine content in 13 D. hansenii strains. h Putrescine; j Spermidine; M Spermine. Data represents mean ± standard deviation for three replicates of the same sample (n = 3). Different superscripts in the same column indicate statistically significant differences (p < 0.05).
From this study it can be concluded that D. hansenii strains DhhBCS004 and L2 (isolated from marine water and citrus fruit, respectively) are especially rich in spermidine and spermine. Since polyamines play a crucial role in cell proliferation and differentiation, these D. hansenii strains could be included among potential biological control agents when administered in the diet as additives or probiotics. Further extensive trials with different target animals and a commercial cost-benefit analysis are necessary to establish their suitability for use as a candidate probiotic in disease prevention and/or growth stimulator of farm animals. Acknowledgements We thank Orlando Lugo for technical support in the HPLC assays. The first author received postdoctoral scholarships from the Mexican Consejo Nacional de Ciencia y Tecnología (CONACYT Grant 164653). This work was funded partly by grants from Ministerio de Ciencia e Innovación (AGL2008-05119-C02-01, Spain) and Fundación Séneca of Murcia, Spain (04538/GERM/06). References Andlid, T., Vázquez-Juárez, R., & Gustafsson, L. (1995). Yeast colonising the intestine of rainbowtrout (Salmo gairdneri) and turbot (Scophtalmus maximus). Microbiology Ecology, 30, 321–324. Bardocz, S., Brown, D. S., Grant, G., & Pusztai, A. (1990). Luminal and basolateral polyamine uptake by rat small intestine stimulated to grow by Phaseolus vulgaris lectin phytohaemagglutinin in vivo. Biochemical Biophysica Acta, 1034, 46–52. Bardocz, S., Duguid, Z. J., Brown, D. S., Grant, G., Pusztai, A., White, A., et al. (1995). The importance of dietary polyamines in cell regeneration and growth. British Journal of Nutrition, 73, 819–828. Barnett, J. A., Payne, R. W., & Yarrow, D. (2000). In J. A. Barnett, R. W. Payne, & D. Yarrow (Eds.), Yeasts: characteristics, and identification (3rd ed.). Cambridge: Cambridge University Press. Baston, O., Stroia, A. L., Moise, D., & Barna, O. (2008). Validation study of a HPLC method able to measure biogenic amines in chicken meat. Food Technology, 31, 44–50. Breuer, U., & Harms, H. (2006). Debaryomyces hansenii-an extremophilic yeast with biotechnological potential. Yeast, 23, 415–437. Cohen, S. (1998). A guide to the polyamines. Oxford: Oxford University Press. pp. 1–543. Eisenberg, T., Knauer, H., Schauer, A., Fussi, H., Büttner, S., Ruckenstuhl, C., et al. (2009). Induction of autophagy by spermidine promotes longevity. Nature Cell Biology, 11, 1305–1314.
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