The potential of bifidobacteria as a source of natural folate

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

The potential of bifidobacteria as a source of natural folate M.R. D’Aimmo1, P. Mattarelli2, B. Biavati2, N.G. Carlsson1 and T. Andlid1 1 Department of Chemical and Biological Engineering ⁄ Food Science, Chalmers University of Technology, Gothenburg, Sweden 2 Department of Agroenvironmental Sciences and Technologies, University of Bologna, Bologna, Italy

Keywords bifidobacteria, folate, folic acid, HPLC, probiotic. Correspondence Thomas Andlid, Department of Chemical and Biological Engineering ⁄ Food Science, Chalmers University of Technology, SE - 412 96 Gothenburg, Sweden. E-mail: [email protected]

2011 ⁄ 1834: received 26 October 2011, revised 14 January 2012 and accepted 8 February 2012 doi:10.1111/j.1365-2672.2012.05261.x

Abstract Aims: To screen 19 strains of bifidobacteria for main folate forms composition in synthetic folate-free and complex folate-containing media. Methods and Results: HPLC was used to analyse deconjugated folates extracted from bacterial biomass. Most strains had a total folate content above 4000 lg per 100 g dry matter (DM). The highest value of 9295 lg per 100 g DM was found in Bifidobacterium catenulatum ATCC 27539 and the lowest in Bifidobacterium animalis ssp. animalis ATCC 25527 containing 220 lg per 100 g DM. Ten strains grew in a synthetic folate-free medium (FFM), showing folate autotrophy and suggesting folate auxotrophy of the remaining nine. In the autotrophic strains, a consistently higher folate level was found in FFM as compared to a more complex folate-containing medium, suggesting reduced requirements for folates in the presence of growth factors otherwise requiring folates for synthesis. The contents of total folate, 5-CH3-H4folate and H4folate were strain dependent. 5-CH3-H4folate dominated in most strains. Conclusions: Our results show that bifidobacteria folate content and composition is dynamic, is strain specific and depends on the medium. Suitable selection of the growth conditions can result in high levels of folate per cell unit biomass. Significance and Impact of the Study: This suggests that certain bifidobacteria may contribute to the folate intake, either directly in foods, such as fermented dairy products, or in the intestine as folate-trophic probiotics or part of the natural microbiota.

Introduction Folate (folic acid; vitamin B9) is a water-soluble vitamin essential for methylation and synthesis of nucleic acids, certain amino acids and proteins necessary for replication and growth (Jacob 2000; Lucock 2000). Folate deficiency is associated with increased risk for malformations in early embryonic brain and spinal cord development (neural tube defects, NTDs) (Botto et al. 1999) and megaloblastic anaemia (Wickramasinghe 2006). Data also suggest increased risk for some forms of cancer (e.g. colon, breast and bladder) (Choi and Mason 2000), Alzheimer’s disease (Clarke et al. 1998) and cardiovascular disease as a result of insufficient folate status (Institute of Medicine, 1998).

In contrast to many micro-organisms and plants, humans are not able to synthesize folate de novo and have to rely on exogenous sources of the vitamin (Sarma et al. 1995). The recommended daily intake of folate, by for instance the Nordic Nutrition Recommendations (Becker et al. 2004), is 300 lg for men, 400 lg for women and 500 lg for lactating or pregnant women (McPartlin et al. 1999). These levels, however, are not easy to reach. Foods rich in folate are, for example, green leafy vegetables like spinach and broccoli, beans and fruits such as oranges, yeast and liver (Eitenmiller and Landen 1999). Naturally, the fraction of such products in the diet depends on availability, economy and eating habits. Moreover, folate in nature is present in different forms and matrixes that vary significantly in stability and bioavailability (Gregory 1996).

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The overall picture is that folate shortage is a common phenomenon, especially in developing countries, poor population segments and women at childbearing age (Antony 2001). Recommendations by health organizations have led to fortification programmes in many countries (Wright et al. 2001). For instance, USA (US Food and Drug Administration, 1996), Canada (Health Canada, 1997) and Chile (Freire et al. 2000) have mandatory fortification of flour and uncooked cereal-grain products, whereas voluntary folic acid fortification of specified foods is implemented in Australia (Metz et al. 2002). Other countries, however, have chosen not to fortify because of the potential link between high doses of synthetic folic acid and the development and progression of certain cancer forms (Mason et al. 2007; Hirsch et al. 2009), as well as masking of vitamin B12 deficiency and thereby the risk of neuropathy (Asrar and O’Connor 2005). Natural folates, in contrast to synthetic folic acid, do not mask vitamin B12 deficiency (Kim et al. 2004) and are probably of lesser risk with respect to overdosing and cancer (Gutstein et al. 1973; Mason 2002; Kim et al. 2004). Therefore, biofortification with natural folates produced by selected micro-organisms may be an alternative to fortification with synthetic folic acid. Some micro-organisms important for fermented food and ⁄ or gut flora, such as yeast and bifidobacteria, can synthesize folate de novo (Klipstein and Samloff 1966; Deguchi et al. 1985; Camilo et al. 1996; Hjortmo et al. 2005). This means that live micro-organisms in food as well as in the intestinal biota may contribute to the human folate intake. It has recently been demonstrated that a portion of folate may be absorbed across the large intestine of both humans (Strozzi and Mogna 2008; Aufreiter et al. 2009) and animals (Kim et al. 2004). In the large intestine, the folate absorption rate is slower than in the small intestine (Wright et al. 2003, 2005); however, the transit time is longer and the rich microbiota probably contributes to a continuous production and hence a more stable folate level than in the small intestine (Aufreiter et al. 2009). Bifidobacteria represent one major group of intestinal bacteria in humans and are often screened for probiotic properties and added to different kinds of dairy and pharmaceutical products (Scardovi 1986). Many strains have been found to produce folate, and information about accumulated cellular and secreted levels is presented in a few studies (Lin and Young 2000; Crittenden et al. 2003; Pompei et al. 2007a,b; Strozzi and Mogna 2008). However, further studies on bifidobacteria folates are motivated for a number of reasons. First, the impact of medium richness (growth factors) has been found crucial for the folate level in yeast. This has not been much studied in bifidobacteria. Second, the relative composition 976

of main folate forms is not known. From yeast studies, it is evident that presence or absence of a certain growth factors as well as specific growth rate affects the level of a specific folate form, which may guide in bioprocessing for higher levels. Knowledge about main forms is also relevant for stability and availability reasons (folate forms vary in stability and bioavailability). Third, previous studies have shown that strains differ much with respect to folates. Therefore, studies on more strains may select potentially useful strains and, for instance, suggest trends for species. Fourth, as different methods for folate extraction and analysis may yield differences in folate data, it is important to compare methods. This far, the microbiological assay has mainly been used for folates in bifidobacteria; here, we use HPLC. The aim of the present work was to investigate the folate levels and main folate form composition in bifidobacteria cultured in vitro. The impact of medium composition on biomass-specific cellular folate content and composition was addressed. We screened different species and strains of bifidobacteria originating from humans, different animals and a fermented product, with a validated HPLC method. Materials and methods Bacterial strains and culturing media A total of 19 Bifidobacterium strains were investigated for their capacity to produce folate. The strains were obtained from Bologna University Scardovi Collection of Bifidobacteria and from ATCC or DSMZ Collections (Table 1). Prior to proceed to the analysis, all the strains of Bifidobacterium spp., provided as freeze-dried cultures, were subcultured in ‘trypticase–phytone–yeast extract’ broth (TPY) (Biavati and Mattarelli 2006), for 24 h, at 37C and anaerobically (Anaerocult A; Merck, Solna, Sweden). Two different culturing media were used for the experiments: (i) TPY, containing trypticase peptone (10 g l)1), phytone peptone (5 g l)1), glucose (15 g l)1), yeast extract (2Æ5 g l)1), Tween 80 (1 ml l)1), cysteine hydrochloride (0Æ5 g l)1), di-potassium hydrogen phosphate (2 g l)1) and magnesium chloride-hexahydrate (0Æ5 g l)1); (ii) synthetic medium developed as ‘folate-free medium’ (FFM) from the ‘Medium D’ (MD) described by Modesto et al. (2003) with modifications mostly consisting of the elimination of sources of folate and addition of a trace metals solution. FFM contained the following components: glucose 15 g l)1, sodium acetate 10 g l)1, ammonium sulfate 10 g l)1, di-potassium hydrogen phosphate 5 g l)1, dihydrogen phosphate 3 g l)1, urea 2 g l)1, ascorbic acid 10 g l)1, Tween 80 1 ml l)1, minerals (MgSO4Æ7H2O 0Æ2 g l)1, FeCl2Æ4H2O 10 mg l)1, MnSO4Æ4H2O 8 mg l)1,

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Bifidobacteria as a source of folate

Table 1 Species and source of 19 Bifidobacterium strains screened for the ability to produce folate Strains

Species

Collection of bifidobacteria*

Bifidobacterium adolescentis

Bifidobacterium bifidum

Bifidobacterium breve Bifidobacterium catenulatum Bifidobacterium longum ssp. infantis Bifidobacterium longum ssp. longum

Bifidobacterium pseudocatenulatum Bifidobacterium animalis ssp. animalis

ATCC ⁄ DSMZ

Source

T

Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces

ATCC 15703 ATCC 15706 B B B B B

5005 1760 2009 2531 1501 ATCC 27539T

B B B B B B B

2130 1860 1954 1990 2160 2327 1280 ATCC 25527T DSMZ 10140T

Bifidobacterium animalis ssp. lactis P 17 Ra 23

References of of of of of of of of of of of of of of of of

adult adult adult infant infant infant infant adult infant infant infant infant infant infant infant rat

Fermented milk Faeces of chicken Faeces of rabbit

Reuter (1963), Scardovi et al. (1971) Reuter (1963), Scardovi et al. (1971) Biavati et al. (1986) Scardovi et al. (1979) Scardovi et al. (1979) Scardovi et al. (1979) Scardovi et al. (1979) Scardovi and Crociani (1974) Scardovi et al. (1979) Scardovi et al. (1979) Scardovi et al. (1979) Scardovi et al. (1979) Scardovi et al. (1979) Scardovi et al. (1979) Scardovi et al. (1979) Scardovi and Trovatelli (1974), Masco et al. (2004) Meile et al. (1997), Masco et al. (2004) Scardovi et al. (1979) Scardovi et al. (1979)

T, type strain. *Received from Bologna University Scardovi Collection of Bifidobacteria. Deposited in the collection.

NaCl 10 mg l)1), trace metals (EDTA 30 mg l)1, CaCl2Æ2H2O 9 mg l)1, ZnSO4Æ7H2O 9 mg l)1, FeSO4Æ7H2O 6 mg l)1, H3BO3 2 mg l)1, MnCl2Æ2H2O 1Æ2 mg l)1, Na2MoO4Æ2H2O 0Æ8 mg l)1, CoCl2Æ2H2O 0Æ6 mg l)1, CuSO4Æ5H2O 0Æ6 mg l)1, KI 0Æ2 mg l)1), vitamins (pyridoxamine pantothenate-HCl 2 mg l)1, nicotinic acid 2 mg l)1, thiamine 2 mg l)1, calcium pantothenate 1 mg l)1, riboflavin 1 mg l)1, p-amino-benzoic acid 50 lg l)1, biotin 50 lg l)1), aminoacids (cysteine-HCl 0Æ5 g l)1, l-alanine 0Æ2 g l)1, dl-arginine 0Æ2 g l)1, dl-asparagine 0Æ2 g l)1 l-aspartic acid 0Æ2 g l)1, glycine 0Æ2 g l)1, l-histidine 0Æ2 g l)1, l-glutamic acid 1 g l)1, lisoleucine 0Æ1 g l)1, l-lysine 0Æ1 g l)1, l-leucine 0Æ2 g l)1, l-methionine 0Æ2 g l)1, dl-phenylalanine 0Æ2 g l)1, l-proline 0Æ2 g l)1, dl-serine 0Æ2 g l)1 l-threonine 0Æ2 g l)1, dl-tyrosine 0Æ2 g l)1, l-trytophan 0Æ2 g l)1, dlvaline 0Æ2 g l)1). The pH of the medium was adjusted to 6Æ6–6Æ8 with a 4 mol l)1 NaOH solution. Both media were autoclaved at 121C for 15 min. Trace metals and glucose were autoclaved separately. Ascorbic acid and the different solutions of minerals, vitamins and amino acids were filter sterilized through a 0Æ22-lm-pore-size filter. Glucose, sodium acetate, ammonium sulfate and magnesium chloride-hexahydrate were purchased from Schar-

lau Chemie, S.A. (Barcelona, Spain). Phytone and yeast extract were purchased from Becton Dickinson and Company (Franklin Lakes, NJ, USA). All other chemicals were obtained from Sigma-Aldrich (Stockholm, Sweden). Culture conditions For the screening in FFM, all the strains were subcultured in broth at 37C anaerobically (Anaerocult A; Merck). Therefore, the strains able to grow for five completed growth cycles were subjected to the analysis. The experiments were conducted in both media, at least in duplicate. Bifidobacterial precultures were cultivated anaerobically (Anaerocult A; Merck), overnight, at 37C in the respective broth. The steps of preparation of medium, bacterial inoculation and cultivation were performed using a Hungate technique (Hungate 1969) modified according to the methods as described by Talwalkar et al. (2001). The medium contained in 15-ml pirex tubes was insufflated with sterile nitrogen gas during boiling to create anaerobic conditions. To preserve anoxic condition, the tubes were sealed with a rubber stopper immediately after the cooling process. Resazurin (2 mg l)1; Sigma-Aldrich) was added into the medium as redox indicator dye.

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Once tubes were ready, precultures were inoculated with a sterile syringe to an initial optical density (OD610) of 0Æ2 and incubated at 37C. Bifidobacterial cultures were collected in the late exponential phase of growth. This stage was normally reached at OD about 1Æ0 (on average 6–8 h) (Lin and Young 2000). For Bifidobacterium breve B 1501 and Bifidobacterium longum ssp. longum B 2160, we assessed folates as a function of time after inoculation in FFM batch culture. From the moment bifidobacteria reached OD = 1Æ0, samples were collected at intervals for HPLC analysis. The bacterial cells were collected by centrifugation (6000 g, 4C, 15 min) and washed twice with cold 0Æ9% NaCl. The pellet was stored in the freezer ()80C) and, when deeply frozen, freeze-dried for 2 days. Folate analysis by HPLC Concentrations of intracellular folate were determined by a validated high-performance liquid chromatography (HPLC) method (Patring et al. 2005). During folates extraction, samples were protected from light, extracted under a nitrogen atmosphere and stored on ice. The samples were analysed in duplicates. Cell extracts were prepared as follows: 0Æ025 g of freeze-dried cells were added to 25 ml of a freshly prepared 0Æ1 mol l)1 phosphate buffer (pH 6Æ1) containing 2% ascorbic acid and 0Æ1% 2,3-dimercapto-1-propanol (Patring et al. 2005). The suspended cells were boiled for 12 min in a water bath and cooled on ice. The supernatant was recovered by centrifugation (27 000 g, 15 min, 4C) and stored in the freezer ()80C) until deconjugation. Rat serum (Scanbur, Uppsala, Sweden) was dialysed in 0Æ1 mol l)1 phosphate buffer containing 0Æ1% 2,3-dimercapto-1-propanol, at 4C during stirring in dialysis tube (cut off 12 000– 14 000 Da) for 3 h. The buffer was changed three times. Deconjugation of folate polyglutamates to monoglutamates was performed by adding 50 ll of the dialysed rat serum to 1 ml of extracted sample in a glass tube. This solution was incubated on a shaking water bath at 37C for 3 h. Rat serum deconjugase enzymes were inactivated by boiling the extracts in a water bath for 5 min. After cooling on ice, the samples were centrifuged (27 000 g, 10 min, 4C) and supernatants were analysed by HPLC. Quantification of folates in TPY was performed in 10 ml of freeze-dried medium. The methodology used for the extraction was the same as described above. The supernatant was purified with SAX-HPLC column prior to the HPLC analysis. (6S)-5,6,7,8-Tetrahydrofolate sodium salt (H4folate) and (6S)-5-CH3-5,6,7,8-tetrahydrofolate sodium salt (5-CH3-H4folate) were used as references for HPLC (Merck Eprova AG, Schaffhausen, Switzerland). Folic acid 978

was added as a reference when performing quantification of folates in TPY. The purity of all standards was checked according to the procedure of Van den Berg et al. (1994) using molar extinction coefficients reported by Eitenmiller and Landen (1999). The concentration of all standard stock solutions was corrected for purity. The HPLC system consisted of a gradient quaternary pump (Jasco PU-2089 plus; Jasco, Mo¨lndal, Sweden), a cooled autosampler (8C) (Jasco AS-2057 plus), a UV detector (Chrompack) and a fluorescence (Jasco FP-920). Individual forms of folate were detected by UV (290 nm) and fluorescence detector (excitation 290 nm, emission 360 nm), and the software jasco chrompass was used as for controlling the HPLC system and for processing the data. The analytical column was Aquasil C18 150 mm · 4Æ6 mm, 3 lm (Thermo Electron Corp., Va¨stra Fro¨lunda, Sweden) and the mobile phase consisted of 30 mmol l)1 phosphate buffer (pH 2Æ3) and acetonitrile. The acetonitrile gradient started at 6% for 5 min, thereafter increasing linearly to 25% in 20 min followed by an increase to 45% in 5 min, which was kept for another 5 min, finally back to the 6% in 1 min. The injection volume was 20 ll, and flow rate was 0Æ4 ml min)1. Statistical analysis We used GLM ancova in spss 15.5 (SPSS Inc., Chicago, IL) to test for effects of the culture medium (TPY or FFM, fixed factor) and strain (random factor) on the dependent variable folate content (total, 5-CH3-H4folate or H4folate). Growth rate was added as a covariate potentially affecting folate content. To increase statistical power, higher order interaction effects were removed from the model when not significant. All continuous variables were tested for normal distribution using Kolmogorov–Smirnov tests, and no variable deviated from expectations. Differences were considered significant at P < 0Æ05. Throughout the manuscript, bacterial folate content is expressed as mean ± the minimum and maximum values from two or three independent experiments ((MAX– MIN) ⁄ 2). Results Folate content in bifidobacteria cultured in TPY The results of the screening in complex medium are presented in Fig. 1, and details about the including strains are described in Table 1. The data show two main things: (i) the content of folate in bifidobacteria is highly strain dependent (Table 2a, b and c) and varies extensively between strains, and (ii) the folate form 5-CH3-H4folate dominates in most strains.

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00 80

00 70

00 60

00 50

00 40

00 30

00 20

10

00

Bif. animalis ssp. animalis ATCC 25527 Bif. animalis ssp. lactis Ra23 Bif. animalis ssp. lactis P17 Bif. animalis ssp. lactis DSMZ10140 Bif. breve 1501 Bif. longum ssp. longum 2160 Bif. longum ssp. infantis 1860 Bif. catenulatum 2130 Bif. longum ssp. infantis 1954 Bif. bifidum 1760 Bif. adolescentis 5005 Bif. longum ssp. longum 2327 Bif. catenulatum ATCC 27539 Bif. adolescentis ATCC 15706 Bif. bifidum 2009 Bif. pseudocatenulatum 1280 Bif. bifidum 2531 Bif. longum ssp. longum 1990 Bif. adolescentis ATCC 15703 0

Strain

M.R. D’Aimmo et al.

Folate content (µg/100 g dry matter) Figure 1 Intracellular biomass-specific folate content and composition in different bifidobacteria cultured in TPY. The strains were harvested at late exponential phase (c. OD = 1). Error bars indicate the minimum and maximum values from two or three independent experiments (mean ± (MAX–MIN) ⁄ 2). Samples from each experiment were independently extracted and analysed twice by HPLC. ( ) Total; ( ) 5-CH3-H4folate and (h) H4folate.

Table 2 ANCOVA results showing the association of quantities of different folate forms (total, H4folate, 5-CH3-H4folate) with culturing medium, bifidobacteria strain and growth rate

(a) Total folate

(b) H4folate

(c) 5-CH3-H4folate

Effects

df*

F

P

Medium Strain Growth rate Medium Strain Growth rate Medium Strain Growth rate

1,35 18,35 1,35 1,35 18,35 1,35 1,35 18,35 1,35

39Æ88 12Æ75 6Æ47 57Æ02 4Æ32 0Æ26 12Æ55 19Æ16 9Æ55