International Journal of Food Sciences and Nutrition
ISSN: 0963-7486 (Print) 1465-3478 (Online) Journal homepage: http://www.tandfonline.com/loi/iijf20
Isoflavone metabolism by a collection of lactic acid bacteria and bifidobacteria with biotechnological interest Pilar Gaya, Ángela Peirotén, Margarita Medina & José Maria Landete To cite this article: Pilar Gaya, Ángela Peirotén, Margarita Medina & José Maria Landete (2016): Isoflavone metabolism by a collection of lactic acid bacteria and bifidobacteria with biotechnological interest, International Journal of Food Sciences and Nutrition, DOI: 10.3109/09637486.2016.1144724 To link to this article: http://dx.doi.org/10.3109/09637486.2016.1144724
Published online: 16 Feb 2016.
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Date: 16 February 2016, At: 23:14
INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION, 2016 http://dx.doi.org/10.3109/09637486.2016.1144724
RESEARCH ARTICLE
Isoflavone metabolism by a collection of lactic acid bacteria and bifidobacteria with biotechnological interest Pilar Gaya, A´ngela Peirote´n, Margarita Medina and Jose´ Maria Landete
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Departamento De Tecnologı´a De Alimentos, Instituto Nacional De Investigacio´n Y Tecnologı´a Agraria Y Alimentaria (INIA), Madrid, Spain
ABSTRACT
ARTICLE HISTORY
Almost all soy isoflavones exist as glycosides, daidzin, genistin, and glycitin. We analyzed the capacity of 92 strains of lactic acid bacteria (LAB) and bifidobacteria with biotechnological interest to process the glycosylated isoflavones daidzin, genistin, and glycitin in their more bioavailable aglycones and their metabolites as dihydrodaidzein (DHD), O-desmethylangolensin, and equol. Representative strains of the four genera studied Lactobacillus, Enterococcus, Lactococcus, and Bifidobacterium were able to produce daidzein, genistein, and glycitein, with the exception of the lactobacilli, which did not produced glycitein in soy extracts. The production of the aglycone isoflavones could be correlated with the -glucosidase activity of the strains. The isoflavone metabolism is limited to the glycoside hydrolysis in the most of these strains. Moreover, Enterococcus faecalis INIA P333 and Lactobacillus rhamnosus INIA P540 were able to transform daidzein in DHD. LAB and bifidobacteria studied in the present work have a great potential in the metabolism of isoflavones and could be selected for the development of functional fermented soy foods.
Received 16 November 2015 Revised 23 December 2015 Accepted 13 January 2016 Published online 12 February 2016
Introduction Isoflavones are flavonoids present in various plants, particularly in soybean germ. They are classified as phytoestrogens since their structures resemble that of estrogen and have a weak affinity for the estrogen receptor (Vaya & Tamir, 2004). Consumption of isoflavones in the form of soy products or supplements is associated with protective effect against cancer (Fournier et al., 1998; Messina et al., 2006), cardiovascular disease (Erdman, 2000), osteoporosis (Messina et al., 2001) and menopausal symptoms (Han et al., 2002). The chemical forms in which phytoestrogens are consumed have an influence in their bioavailability and therefore in their antioxidant and estrogenic/antiestrogenic activities (Landete et al., 2015). In soy and unfermented soy foods almost all isoflavones exist as glycosides, being daidzin and genistin the most abundant. Isoflavone glycosides are less estrogenic than their respective aglycones, and they are not absorbed by the human intestine because of their higher hydrophilicity and molecular weights (Hur et al., 2000). Their bioavailability requires the conversion of glycosides to aglycones (Figures 1 and 2) via the action of -glycosidase from gut bacteria that colonize the small and the large intestine (Setchell et al., 2002a, b). Glycoside isoflavones could be
KEYWORDS
-glucosidase; bifidobacteria; dihydrodaidzein; isoflavones; lactic acid bacteria
also transformed to a lesser extent by endogenous enzymes in the small intestine (Richelle et al., 2002). Aglycone isoflavones can then be absorbed through the gut epithelium (Decroos et al., 2005) or be subjected to further metabolism by the intestinal microbiota (Decroos et al., 2005; Setchell & Cassidy, 1999; Zubik & Meydani, 2003). Thus, daidzein can be converted via dihydrodaidzein (DHD) to O-desmethylangolensin (O-DMA) or equol by enzymes of intestinal bacteria (Figure 2). The extent of this metabolism appears to be highly variable among individuals due to differences in the gut microbiota and is influenced by other components of the diet (Setchell & Cassidy, 1999). In this work we analyze the capacity of Lactococcus, Enterococcus, Lactococcus, and Bifidobacterium strains with biotechnological interest (Rodrı´guez et al., 2003, 2012) to metabolize the glycosylated isoflavones daidzin, genistin, and glycitin into their aglycones and secondary metabolites as DHD, O-DMA, and equol.
Material and methods Bacterial growth conditions Lactic acid bacteria (LAB) and bifidobacteria strains tested in this study are listed in Tables 1–4. All isolates
CONTACT Jose´ Maria Landete [email protected] Departamento de Tecnologia de Alimentos, Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA), Carretera de La Coruna Km 7.5, Madrid 28040, Spain ! 2016 Taylor & Francis
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Figure 1. Molecular structure of glycitin and genistin and theirs aglycones glycitein and genistein.
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Table 1. Production of daidzein, genistein, glycitein, dihydrodaidzein (DHD), and -glucosidase activity by Lactobacillus strains with biotechnological interest. Strains L. paracasei INIA P55 L. paracasei INIA P74 Lactobacillus sp. INIA P143 Lactobacillus sp. INIA P160 L. paracasei INIA P773 L. rhamnosus INIA P224 L. salivarius INIA P235 L. rhamnosus INIA P334 L. rhamnosus INIA P374 L. paracasei INIA P393 L. paracasei INIA P461 L. rhamnosus INIA P535 L. rhamnosus INIA P540 L. rhamnosus INIA P556 L. reuteri INIA P568 L. reuteri INIA P574 L. reuteri INIA P580 L. reuteri INIA P581 L. reuteri INIA P582 L. rhamnosus INIA P603 L. paracasei INIA P651 L. paracasei INIA P655 L. paracasei INIA P708
Figure 2. Metabolic pathway of equol.
belong to the INIA culture collection, they were selected from INIA collection for its biotechnological and probiotic properties (Rodrı´guez et al., 2003, 2012). Lactobacillus, Enterococcus, and Bifidobacterium strains were isolated from mother milk and infant feces the healthy infants, with the exception of Lactobacillus reuteri strains which were isolated from pig feces.
Daidzein Genistein Glycitein DHD +
+
+
+ +
-glucosidase activity
+
+ + + +
+
+
+
+
+
+ + +
+
+ + +
+ + + + +
Lactococcus strains were isolated from dairy products as raw milk or cheese. Lactobacillus and Enterococcus were routinely cultivated under anaerobic conditions at 37 C in MRS broth (Oxoid, Ltd. Basingstoke, Hampshire, England). Lactococcus were grown at 30 C in M17 broth (BD, Le Pont de Claix, France) supplemented with glucose (5 g/L) (Sigma Aldrich, Poole, Dorset, UK). Bifidobacterium were cultivated under strict anaerobic conditions at 37 C in RCM broth (BD). Slackia isoflavoniconvertens DSM22006, used as equol-positive control, was grown in Wilkins-Chalgren anaerobe broth (Oxoid) at 37 C under strict anaerobic conditions.
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Table 2. Production of daidzein, genistein, glycitein, dihydrodaidzein (DHD), and -glucosidase activity by Enterococcus strains with biotechnological interest.
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Strains E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E.
Daidzein Genistein Glycitein DHD
faecium INIA P1 faecalis INIA P8 faecalis INIA P14 faecalis INIA P23 faecalis INIA P35 faecalis INIA P52 faecalis INIA P56 faecalis INIA P90 faecium INIA P125 faecalis INIA P127 faecalis INIA P236 faecalis INIA P290 faecalis INIA P333 faecalis INIA P419 faecalis INIA P429 faecium INIA P445 faecium INIA P545 faecium INIA P455 faecalis INIA P464 faecalis INIA P474 faecium INIA P552 faecium INIA P553 faecalis INIA P562
+
+
-glucosidase activity
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
+
+
+ +
+
+ +
+
+ +
Table 3. Production of daidzein, genistein, glycitein, dihydrodaidzein (DHD), and -glucosidase activity by Lactococcus strains with biotechnological interest. Strains L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis
Daidzein Genistein Glycitein DHD cremoris ZA24 diacetylactis ZA37 diacetylactis ZA57 diacetylactis ZA86 diacetylactis SJ46 lactis ESI718 lactis TAB26 lactis TAB75 cremoris BO2 diacetylactis BO14 cremoris BO68 diacetylactis BO112 diacetylactis BO128 INIA13 INIA14 INIA15 lactis INIA415 lactis INIA437 cremoris INIA450 lactis ESI153 lactis ESI240 lactis ESI277 lactis ESI515
-glucosidase activity
+
+ + +
+ +
+ + +
+ + +
+ + + + + +
+ + +
+
+
+
+ + + +
+ +
+
Metabolism assay of soybean extracts by LAB and bifidobacteria LAB and bifidobacteria strains were inoculated in 10 mL of BHI medium (Biolife, Milan, Italy) containing 0.5 g/L of l-cysteine supplemented with the soybean extract (4 g/L) (SoyLifes EXTRA; Frutarom Netherlands BV, Veenendaal, the Netherlands), in DMSO, and the pH was adjusted to 7.4 with NaOH. The amount of DMSO present in the media did not affect bacterial growth.
Table 4. Production of daidzein, genistein, glycitein, dihydrodaidzein (DHD), and -glucosidase activity by bifidobacteria strains with biotechnological interest. Strains B. adolescentis INIA P879 B. pseudolongum INIA P2 B. infantis INIA P737 B. breve INIA P712 B. adolescentis INIA P784 B. animalis INIA P797 B. infantis INIA P718 B. longum INIA P911 B. adolescentis INIA P916 B. pseudolongum INIA P965 B. pseudolongum INIA P120 B. longum INIA P678 B. longum INIA P132 B. bifidum INIA P685 B. breve INIA P730 Bifidobacterium sp. INIA P819 B. dentium INIA P882 B. infantis INIA P731 B. pseudocatenulatum INIA P885 B. catenulatum INIA P836 B. catenulatum INIA P926 Bifidobacterium sp. INIA P996 B. animalis INIA P900
Daidzein Genistein Glycitein DHD + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + +
+ + + + +
+ + + + + + + + +
-glucosidase activity + + + + + + + + + + + + + + + + + + + + + + +
The inoculated broths supplemented with the polyphenols were incubated in sealed jars (Oxoid) under anaerobic conditions using AnaeroGen sachets (Oxoid) at 37 C for 7 d. Non-inoculated BHI with soybean extracts was used as control. Extraction of isoflavones After incubations of the inoculated broths, isoflavones were extracted twice with 2 mL of diethyl ether and twice with 2 mL of ethyl acetate according to Gaya et al. (2016). The solvents were evaporated at room temperature under N2 stream and the residue was dissolved in 300 mL methanol/water (50:50, v/v). Filtering through a 0.22 mm cellulose acetate filter (Millipore, Madrid, Spain) was performed before transferring the extracts to HPLC vials and storing them at 20 C. HPLC-PAD analysis Analysis was carried out on a HPLC-PAD Beckman System Gold (Beckman Coulter Inc., Fullerton, CA), comprising an autosampler module 508, binary pump module 126, diode array detector module 168, and 32 Karat Software chromatography manager. Separation of phenolic compounds was achieved on a reverse phase Nova-Pak C18 column (300 3.9 mm, 4 mm) (Waters, Barcelona, Spain). The analytical conditions were based on those described by Gaya et al. (2016). A gradient consisting of solvent A (water/acetic acid, 98:2 v/v) and solvent B
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(water/acetonitrile/acetic acid, 78:20:2 v/v/v) was applied at a flow rate of 1 mL/min from the beginning to 55 min, and 1.2 mL/min from this point to the end. The gradient profile was 0–55 min, 100%–20% A; 55–70 min, 20%– 10% A; 70–80 min, 10%–5% A; 80–110 min, 100% B. Detection was performed by scanning from 210 nm to 400 nm with an acquisition speed of 1 s. A volume of 25 mL was injected. The solvents used, methanol, acetic acid, and acetonitrile were of HPLC grade (LabScan, Gliwice, Poland).
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Identification and quantification of phenolic compounds Chromatographic peaks were identified previously by HPLC-ESI/MS and confirmed by comparison of retention times and characteristics of UV spectra with those of standards (Gaya et al., 2016). The HPLC grade standard compounds daidzein, daidzin, glycitein, equol, genistein, genistin, and glycitin were purchased from LC Laboratories (New Boston Street Woburn, MA). DHD was purchased from Toronto Research Chemicals (TRC) (Toronto, Ontario, Canada). Quantification was made using the external standard calibration curves, with commercial standards (Gaya et al., 2016). Stock solutions of phytoestrogens were prepared in DMSO (Sigma-Aldrich) in a concentration of 10 mg/L. Initial concentrations of daidzein, genistein, and glicitein in control samples were subtracted for calculating the production of these metabolites by the strains analyzed. DHD was not detected in control samples. b-glucosidase activity assays Bacterial cultures were centrifuged at 4000g for 3 min and resuspended in phosphate buffer (0.2 M, pH 6.8). Suspensions were then incubated aerobically for 30 min at 37 C with p-nitrophenyl- -D-glucopyranoside (2.5 mM) (Sigma-Aldrich, St Louis, MO). Release of pnitrophenol was measured with a spectrophotometer (Beckman DU 650 Fullerton, CA) at 405nm before and after incubation (Berg et al., 1978).
Results Isoflavone metabolism was analyzed in 92 strains of bacteria including 23 isolates of each of the genera Lactobacillus, Enterococcus, Lactococcus, and Bifidobacterium. Results of the HPLC analysis of the isoflavone metabolism are listed in Tables 1–4. Fiftythree of the isolates (57.7%) produced at least one of the aglycones studied. Production of daidzein, genistein, and
glycitein from daidzin, genistin, and glycitin in soy extracts was observed in strains of the four genera studied, with the exception of the Lactobacillus genus which did not show any glycitein producer strain. Bifidobacteria were the group showing the greatest ability to metabolize isoflavones since all of Bifidobacterium strains produced at least one compound. Among the other three genera the percentage of strains producing at least one of them was 39.1%, 43.5%, and 47.8% for Lactobacillus, Enterococcus, and Lactococcus respectively. For its part, the isoflavone genistein was the most frequently transformed into its aglycone by all groups of bacteria analyzed and glycitein the least. While production of genistein was observed as the sole compound detected in some isolates, daidzein and glycitein were never detected alone. The analysis of the results reveals that when isoflavone glycosides in soy extracts are converted into their aglycones, genistein is produced. So, production of daidzein or glycitein is always accompanied by the production of genistein in soy extracts. This does not occur in reverse. Then, LAB and bifidobacteria show higher affinity for the transformation of genistin in genistein in soy extracts that the rest of deglycosylations, although the initial concentration of daidzin is much higher. Moreover, the range of production of the isoflavone aglycones was highly variable between strains. Nine Lactobacillus strains produced genistein and only 3 strains produced daidzein; none of the Lactobacillus strains tested produced glycitein (Table 1). Daidzein production from Lactobacillus ranged from 87.67 to 135.64 mg/L and genistein from 38.90 to 209.49 mg/L. The analysis of the metabolism of isoflavones by Enterococcus showed that 9 out of 23 and 10 out of 23 strains were able to produce daidzein and genistein, respectively. Only 4 strains showed glycitein production (Table 2). Daidzein production from Enterococcus ranged from 22.38 to 136.44 mg/L, genistein from 28.25 to 93.53 mg/L and glycitein between 65.98 and 84.58 mg/L. Only one Lactococcus strain was able to produce daidzein, although 11 out of the 23 strains tested produced genistein and two strains glycitein (Table 3). Daidzein production from L. lactis ESI515 was 210.23 mg/L, genistein production by Lactococcus strains ranged from 67.05 to 208.78 mg/L and glycitein from 105.01 to 146.89 mg/L. All the bifidobacteria analyzed produced genistein, and daidzein and glycitein were produced by 78.3% and 60.9% strains, respectively (Table 5). Daidzein production by bifidobacteria ranged from 44.94 to 261.74 mg/L, genistein from 9.47 to 268.99 mg/L, and glycitein from 47.03 to 130.78 mg/L.
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Initial concentrations of daidzin (665 mg/L), genistin (412 mg/L), and glycitin (214 mg/L) were reduced by the Lactobacillus, Enterococcus, Lactococcus, and Bifidobacterium producing their respective aglycones. Even, some strains completely consumed these initial compounds as L. lactis ESI515. Equol or O-DMA producing strains were not found in the present work, although equol production was observed when S. isoflavoniconvertens DSM22006 was incubated with soy extracts. The precursor metabolite of equol DHD was detected in Lactobacillus rhamnosus INIA P540 (1.09 mg/L) and Enterococcus faecalis INIA P333 (0.87 mg/L). Moreover, E. faecalis INIA P333 was able to produce daidzein, genistein, and glycitein (Figure 3). Fifteen out of 92 isolates (16.3%) were capable of producing glycitein, dadzein, and genistein. Ten Bifidobacterium and four Enterococcus strains produced the three aglycones. Whereas only one of the Lactococcus strain produced the three aglycones. Results from all the bacteria strains studied are summarized in Table 5. -glucosidase enzyme can transform daidzin, genistin, and glycitin into daidzein, genistein, and glycitein,
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respectively, as we observed when LAB and bifidobacteria with -glucosidase activity were incubated in the presence of daidzin, genistin, and glycitin (data not shown). However, results with soy extracts indicate a greater affinity for genistein. -glucosidase activity was confirmed (Tables 1–4) in all the strains able to transform their aglycones by means of the colorimetric method assayed. -glucosidase activity with p-nitrophenyl- -D-glucopyranoside was detected in all soy extracts exhibiting deglycosylation of isoflavones. However, deglycosylation of p-nitrophenyl -D-glucopyranoside to yield p-nitrophenol was observed in strains that did not produce the isoflavone deglycosylation in soy extracts. Fifty-three of the isolates (57.7%) produced at least one of the aglycones studied, whereas sixty four of the isolates (69.6%) showed -glucosidase activity. L. rhamnosus INIA P540 exhibited -glucosidase activity but the strain could not transform daidzein, genistein, or glycitin in its aglycone in soy extracts. Analysis with pure compounds daidzin, genistin, and glycitin showed that this strain was able to produce daidzein, genistein, and glycitein (data not shown).
Table 5. Production daidzein, genistein, glycitein, dihydrodaidzein, and -glucosidase activities by 92 Lactobacillus, Enterococcus, Lactococcus, and Bifidobacterium strains. Lactobacillus Enterococcus Lactococcus Bifidobacterium
Strains
Daidzein
Genistein
Glycitein
DHD
-glucosidase activity
23 23 23 23
3 (13.0%) 9 (39.1%) 1 (4.3%) 18 (78.3%)
9 (39.1%) 10 (43.5%) 11 (47.8%) 23 (100%)
0 4 (17.5%) 2 (8.7%) 14 (60.9%)
1 (4.3%) 1 (4.3%) 0 0
14 (60.9%) 10 (43.5%) 17 (73.9%) 23 (100%)
Figure 3. Identification of isoflavones by HPLC-PAD in soybean extracts control (A). (1, daidzin; 2, glycitin; 3, genistin; 4, dihydrodaidzein; 5, daidzein; 6, glycitein; 7, genistein). In soybean extracts incubated with E. faecalis INIA PRO333 (B).
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Discussion The transformation of glycosylated isoflavones, mainly daidzin, to their respective aglycones by individual bacterial strains or mixed cultures has been reported (Cho et al., 2012; Landete et al., 2014; Otieno et al., 2006; Raimondi et al., 2009; Wei et al., 2007). However, this is the first time a study is made on a large collection of LAB and bifidobacteria. Moreover, this work analyzes the capacity of these strains to metabolize the glycosylated isoflavones daidzin, genistin, and glycitin into their aglycones and secondary metabolites as DHD, O-DMA, and equol. The 92 bacterial strains investigated in the present work were selected from INIA collection for its biotechnological and probiotic properties (Rodrı´guez et al., 2003, 2012). It is noteworthy, as although bacteria with -glucosidase activity are capable of producing the deglycosylation of daidzin, genistin, and glycitin, the results of the tables show how the LAB and bifidobacteria have priority in the production of genistein from genistin, and the daidzein and/or glycitein production do not occur in many cases in soy extracts. One possible explanation is that -glucosidase has more affinity for genistein as already shown (Matsuura & Obata, 1993). The transformation of the glycosylated isoflavones in their aglycone is of great interest for three reasons; increases bioavailability (Selma et al., 2009), increased antioxidant activity (Landete et al., 2014), and facilitates the transformation to equol (Wang et al., 2007). The bioavailability of phenolics is rather low (Manach et al., 2005). This bioavailability can be even lower when the food polyphenols have a large molecular weight, as is the case of complex flavonoid conjugates with several sugars. The microbiota metabolizes these complex polyphenols into smaller molecules which are generally better absorbed in the intestine (Selma et al., 2009). Soybean isoflavones exist as glycosides that are not absorbed intact across the enterocyte of healthy adults because of their higher hydrophilicity and molecular weight (Hur et al., 2000). Their bioavailability requires the conversion of glycosides into aglycones via the action of -glucosidase from bacteria (Izumi et al., 2000). According to our results, a high proportion of LAB and bifidobacteria possess -glucosidase activity and are capable of transforming the glycoside isoflavones into their aglycone, and promote their absorption. Improvement of the antioxidant activity of fermented food products containing glycosylated polyphenols have been described previously. Fermentation of soymilk by L. rhamnosus CRL981 enhanced antioxidant activity and increased isoflavone aglycone content (Marazza et al., 2012). Pyo et al. (2005) demonstrated an increment in
the antioxidant activity of fermented soybean due to the augmented content of aglycones. Landete et al. (2014) demonstrated for the first time the association between the deglycosylation of specific aryl glycosides by L. plantarum 748 with a rise in their antioxidant activity. Increased isoflavone aglycone content in fermented soymilk with L. rhamnosus C6 is likely to improve the biological functionality of soymilk (e.g. antioxidant activity, alleviation of hormonal disorders in postmenopausal women, and so forth) (Hati et al., 2015). Then, LAB and bifidobacteria could be used as functional starter culture to increase the antioxidant activity of plant foods during fermentation. Moreover, De Boever et al. (2001) showed as the interactions between the LAB as L. reuteri and the soygerm powder suggesting the interest that it may be interesting to combine both culturesin fermented milk. The soygerm powder may protect L. reuteri against the bile salt toxicity of the small intestine, whereas the fermentation process releases interesting bioactive isoflavones in the food supplement. Soybean extracts and preparations with LAB and bifidobacteria may be regarded as effective natural and functional dietary food supplements due to their remarkable content of bioactive aglycones and to their significant radical scavenging capacity. The use of probiotic microorganisms in fermented soymilk is beneficial to alter the biological isoflavone aglycone and to improve functional benefits for consumers (Wei et al., 2007). The hydrolysis of glycosides results in metabolites that are potentially more biologically active than the parent compounds (Selma et al., 2009). Furthermore, the glucosides are known to be less bioactive than their respective aglycones (Landete et al., 2015). Daidzein is converted to equol by enzymes of intestinal bacteria in the equol-producer individuals. The clinical effectiveness of soy isoflavones may be a function of the ability to biotransform soy isoflavones to the more potent estrogenic metabolite equol, which may enhance the actions of soy isoflavones, owing to its greater affinity for estrogen receptors, unique antiandrogenic properties, and superior antioxidant activity (Setchell, 2004). Equol is absorbed more efficiently through the colon wall than daidzein (Decroos et al., 2005), appears in plasma after intake of daidzein and remains in plasma for a relatively longer period of time than do genistein and daidzein (Zubik & Meydani, 2003). In this work, we did not find equol-producing strains. Equol production has usually been associated with family Coriobactericeae with the exception of a strain of L. garviae (Uchiyama et al., 2007). More recently, the production of equol from daidzein by B. breve 15700 and B. longum BB536 has been demonstrated
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INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION
(Elghali et al., 2012). However, we did not observed equol production by B. longum BB536 in our laboratory (data not shown). An anaerobic incubation mixture of two bacterial strains Eggerthella spp. Julong 732 and Lactobacillus spp. Niu-O16, that individually transformed DHD to S-equol and daidzein to DHD respectively, produced S-equol from daidzein through DHD. The biotransformation kinetics of daidzein by the mixed culture showed that the production of S-equol from daidzein was significantly enhanced, as compared with the production of S-equol from DHD, by Eggerthella sp. Julong 732 alone (Wang et al., 2007). Then, strains capable of transforming isoflavone glycosides and their aglycones and, essentially, strains capable of producing DHD facilitate the production of equol according to our results So, L. rhamnosus INIA P540 and E. faecalis INIA P333 were able to produce DHD. Moreover, E. faecalis INIA P333 was able to produce daidzein, genistein, and glycitein, being considered as a strain of great interest.
Conclusions LAB and bifidobacteria possessing -glucosidase activity are relevant in the production of compounds with more estrogenic/antiestrogenic and antioxidant activities and bioavailability as daidzein, genistein, glycitein and mainly DHD. Strains with biotechnological and probiotic properties could be further selected for the development of functional fermented soy foods. The ability to metabolize isoflavones can be considered a remarkable characteristic of microorganisms with probiotic potential.
Disclosure statement The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Funding information This work was supported by RM2012-00004-00-00. J.M.L. has a postdoctoral contract with the research program ‘‘Ramo´n y Cajal’’ (MINECO, Spain).
References Berg JO, Nord CE, Wadstrom T. 1978. Formation of glycosidases in batch and continuous culture of Bacteroides fragilis. Appl Environ Microbiol 35:269–273. Cho C-W, Jeong H-C, Hong H-D, Kim Y-C, Choi SY, Kim K, Ma JY, Lee Y-C. 2012. Bioconversion of isoflavones during
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the fermentation of samso-eum with Lactobacillus strains. Biotechnol Biopr Engin 17:1062–1067. Decroos K, Vanhemmens S, Cattoir S, Boon N, Verstraete W. 2005. Isolation and characterisation of an equol-producing mixed microbial culture from a human faecal sample and its activity under gastrointestinal conditions. sample its under conditions. Arch Microbiol 183:45–55. De Boever P, Wouters R, Verstraete W. 2001. Combined use of Lactobacillus reuteri and soygerm powder as food supplement. Lett Appl Microbiol 33:420–424. Elghali S, Mustafa S, Amid M, Manap MYABD, Ismail A, Abas F. 2012. Bioconversion of daidzein to equol by Bifidobacterium breve 15700 and Bifidobacterium longum BB536. J Funct Foods 4:736–745. Erdman JW Jr. 2000. AHA Science Advisory: soy protein and cardiovascular disease: a statement for healthcare professionals from the Nutrition Committee of the AHA. Circulation 102:2555–2559. Fournier DB, Erdman JW Jr, Gordon GB. 1998. Soy, its components, and cancer prevention: a review of the in vitro, animal, and human data. Cancer Epidemiol Biomarkers Prev 7:1055–1065. ´ lvarez I, Landete JM. 2016. A Gaya P, Arque´s JL, Medina M, A new HPLC-PAD/HPLC-ESI-MS method for the analysis of phytoestrogens produced by bacterial metabolism. Food Anal Meth 9:537–547. Han KK, Soares JM, Haidar MA, de Lima GR, Baracat E. 2002. Benefits of soy isoflavone therapeutic regimen on menopausal symptoms. Obstet Gynecol 99:389–394. Hati S, Vij S, Pal B, Singh BP, Mandal S. 2015. -Glucosidase activity and bioconversion of isoflavones during fermentation of soymilk. J Sci Food Agric 95:216–220. Hur HG, Lay JO Jr, Beger RD, Freeman JP, Rafii F. 2000. Isolation of human intestinal bacteria metabolizing the natural isoflavone glycosides daidzin and genistin. Arch Microbiol 174:422–428. Izumi T, Piskula MK, Osawa S, Obata A, Tobe K, Saito M, Kataoka S, et al. 2000. Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. J Nutr 130:1695–1699. Landete JM, Arque´s J, Medina M, Gaya P, de La Rivas B, Mun˜oz R. 2015. Bioactivation of phytoestrogens: intestinal bacteria and health. Crit Rev Food Sci Nutr. [Epub ahead of print]. doi:10.1080/10408398.2013.78982. Landete JM, Curiel JA, Rodrı´guez H, de las Rivas B, Mun˜oz R. 2014. Aryl glycosidases from Lactobacillus plantarum increase antioxidant activity of phenolic compounds. J Funct Foods 7:322–329. Manach C, Williamson G, Morand C, Scalbert A, Re´me´sy R. 2005. Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr 81:230S–242S. Marazza JA, Nazareno MA, Savoy de Gion G, Garro MS. 2012. Enhancement of the antioxidant activity capacity of soymilk by fermentation with Lactobacillus rhamnosus. J Funct Foods 4:594–601. Matsuura M, Obata A. 1993. -glucosidases from soybean hydrolyze daidzin and genistin. J Food Sci 58:144–147. Messina M, Gugger ET, Alekel DL. 2001. Soy protein, soybean isoflavones, and bone health: a review of the animal and human data. In: Wildman R, editor. Handbook of nutraceuticals and functional foods. Boca Raton, FL: CRC Press. p 77–98.
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Messina M, Kucuk O, Lampe JW. 2006. An overview of the health effects of isoflavones with an emphasis on prostate cancer risk and prostate-specific antigen levels. emphasis prostate risk prostatespecific antigen. J AOAC Int 89:1121–1134. Otieno DO, Ashton JF, Shah NP. 2006. Evaluation of enzymic potential for biotransformation of isoflavone phytoestrogen in soymilk by Bifidobacterium animalis, Lactobacillus acidophilus and Lactobacillus casei. Food Res Int 39:394–407. Pyo Y-H, Lee T-C, Lee Y-C. 2005. Enrichment of bioactive isoflavones in soymilk fermented with -glucosidase producing lactic acid bacteria. Food Res Int 38:551–559. Raimondi S, Roncaglia L, De Lucia M, Amaretti A, Leonardi A, Pagnoni UM, Rossi M. 2009. Bioconversion of soy isoflavones daidzin and daidzein by Bifidobacterium strains. Appl Microbiol Biotechnol 81:943–950. Richelle M, Priclmore-Merten S, Bodenstab S, Enslen M, Offord EA. 2002. Hydrolysis of isoflavone glycosides to aglycones by -glycosidase does not alter plasma and urine isoflavone pharmacokinetics in postmenopausal women. J Nutr 132:2587–2592. Rodrı´guez E, Arque´s JL, Rodrı´guez R, Nun˜ez M, Medina M. 2003. Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits. Lett Appl Microbiol 37:259–263. Rodrı´guez E, Arque´s JL, Rodrı´guez R, Peiroten A, Landete JM, Medina M. 2012. Antimicrobial properties of probiotic strains isolated from breast-fed infants. J Funct Foods 4:542–551. Selma MV, Espı´n JC, Toma´s-Barberan FA. 2009. Interaction between phenolics and gut microbiota: role in human health. J Agric Food Chem 57:6485–6501.
Setchell KDR. 2004. Equol – origins, actions, and clinical relevance of this specific soy isoflavone metabolite. J Nutr 134:1235S–1236S. Setchell KDR, Brown NM, Lydeking-Olsen E. 2002b. The clinical importance of the metabolite equol-a clue to the effectiveness of soy and its isoflavones – clue the of and isoflavones. J Nutr 132:3577–3584. Setchell KDR, Brown NM, Zimmer-Nechemias L, Brashear WT, Wolfe BE, Kirschner AS, Heubi JE. 2002a. Evidence for lack of absorption of soy isoflavone glycosides in humans, supporting the crucial role of intestinal metabolism for bioavailability. Am J Clin Nutr 76:447–453. Setchell KDR, Cassidy A. 1999. Dietary isoflavones: biological effects and relevance to human health. J Nutr 129:758S–767S. Uchiyama S, Ueno T, Suzuki T. 2007. Identification of a newly isolated equol-producing lactic acid bacterium from the human feces. J Int Microbiol 21:217–220. Vaya J, Tamir S. 2004. The relation between the chemical structure of flavonoids and their estrogen-like activities. Curr Med Chem 11:1333–1343. Wang X-L, Kim H-J, Kang S-I, Kim S-I, Hur H-G. 2007. Production of phytoestrogen S-equol from daidzein in mixed culture of two anaerobic bacteria. Arch Microbiol 187:155–160. Wei Q-K, Chen T-R, Chen J-T. 2007. Using of Lactobacillus and Bifidobacterium to product the isoflavone aglycones in fermented soymilk. Int J Food Microbiol 117:120–124. Zubik L, Meydani M. 2003. Bioavailability of soybean isoflavones from aglycone and glucoside forms in American women. Am J Clin Nutr 77:1459–1465.
ISSN: 0963-7486 (Print) 1465-3478 (Online) Journal homepage: http://www.tandfonline.com/loi/iijf20
Isoflavone metabolism by a collection of lactic acid bacteria and bifidobacteria with biotechnological interest Pilar Gaya, Ángela Peirotén, Margarita Medina & José Maria Landete To cite this article: Pilar Gaya, Ángela Peirotén, Margarita Medina & José Maria Landete (2016): Isoflavone metabolism by a collection of lactic acid bacteria and bifidobacteria with biotechnological interest, International Journal of Food Sciences and Nutrition, DOI: 10.3109/09637486.2016.1144724 To link to this article: http://dx.doi.org/10.3109/09637486.2016.1144724
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Date: 16 February 2016, At: 23:14
INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION, 2016 http://dx.doi.org/10.3109/09637486.2016.1144724
RESEARCH ARTICLE
Isoflavone metabolism by a collection of lactic acid bacteria and bifidobacteria with biotechnological interest Pilar Gaya, A´ngela Peirote´n, Margarita Medina and Jose´ Maria Landete
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Departamento De Tecnologı´a De Alimentos, Instituto Nacional De Investigacio´n Y Tecnologı´a Agraria Y Alimentaria (INIA), Madrid, Spain
ABSTRACT
ARTICLE HISTORY
Almost all soy isoflavones exist as glycosides, daidzin, genistin, and glycitin. We analyzed the capacity of 92 strains of lactic acid bacteria (LAB) and bifidobacteria with biotechnological interest to process the glycosylated isoflavones daidzin, genistin, and glycitin in their more bioavailable aglycones and their metabolites as dihydrodaidzein (DHD), O-desmethylangolensin, and equol. Representative strains of the four genera studied Lactobacillus, Enterococcus, Lactococcus, and Bifidobacterium were able to produce daidzein, genistein, and glycitein, with the exception of the lactobacilli, which did not produced glycitein in soy extracts. The production of the aglycone isoflavones could be correlated with the -glucosidase activity of the strains. The isoflavone metabolism is limited to the glycoside hydrolysis in the most of these strains. Moreover, Enterococcus faecalis INIA P333 and Lactobacillus rhamnosus INIA P540 were able to transform daidzein in DHD. LAB and bifidobacteria studied in the present work have a great potential in the metabolism of isoflavones and could be selected for the development of functional fermented soy foods.
Received 16 November 2015 Revised 23 December 2015 Accepted 13 January 2016 Published online 12 February 2016
Introduction Isoflavones are flavonoids present in various plants, particularly in soybean germ. They are classified as phytoestrogens since their structures resemble that of estrogen and have a weak affinity for the estrogen receptor (Vaya & Tamir, 2004). Consumption of isoflavones in the form of soy products or supplements is associated with protective effect against cancer (Fournier et al., 1998; Messina et al., 2006), cardiovascular disease (Erdman, 2000), osteoporosis (Messina et al., 2001) and menopausal symptoms (Han et al., 2002). The chemical forms in which phytoestrogens are consumed have an influence in their bioavailability and therefore in their antioxidant and estrogenic/antiestrogenic activities (Landete et al., 2015). In soy and unfermented soy foods almost all isoflavones exist as glycosides, being daidzin and genistin the most abundant. Isoflavone glycosides are less estrogenic than their respective aglycones, and they are not absorbed by the human intestine because of their higher hydrophilicity and molecular weights (Hur et al., 2000). Their bioavailability requires the conversion of glycosides to aglycones (Figures 1 and 2) via the action of -glycosidase from gut bacteria that colonize the small and the large intestine (Setchell et al., 2002a, b). Glycoside isoflavones could be
KEYWORDS
-glucosidase; bifidobacteria; dihydrodaidzein; isoflavones; lactic acid bacteria
also transformed to a lesser extent by endogenous enzymes in the small intestine (Richelle et al., 2002). Aglycone isoflavones can then be absorbed through the gut epithelium (Decroos et al., 2005) or be subjected to further metabolism by the intestinal microbiota (Decroos et al., 2005; Setchell & Cassidy, 1999; Zubik & Meydani, 2003). Thus, daidzein can be converted via dihydrodaidzein (DHD) to O-desmethylangolensin (O-DMA) or equol by enzymes of intestinal bacteria (Figure 2). The extent of this metabolism appears to be highly variable among individuals due to differences in the gut microbiota and is influenced by other components of the diet (Setchell & Cassidy, 1999). In this work we analyze the capacity of Lactococcus, Enterococcus, Lactococcus, and Bifidobacterium strains with biotechnological interest (Rodrı´guez et al., 2003, 2012) to metabolize the glycosylated isoflavones daidzin, genistin, and glycitin into their aglycones and secondary metabolites as DHD, O-DMA, and equol.
Material and methods Bacterial growth conditions Lactic acid bacteria (LAB) and bifidobacteria strains tested in this study are listed in Tables 1–4. All isolates
CONTACT Jose´ Maria Landete [email protected] Departamento de Tecnologia de Alimentos, Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA), Carretera de La Coruna Km 7.5, Madrid 28040, Spain ! 2016 Taylor & Francis
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Figure 1. Molecular structure of glycitin and genistin and theirs aglycones glycitein and genistein.
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Table 1. Production of daidzein, genistein, glycitein, dihydrodaidzein (DHD), and -glucosidase activity by Lactobacillus strains with biotechnological interest. Strains L. paracasei INIA P55 L. paracasei INIA P74 Lactobacillus sp. INIA P143 Lactobacillus sp. INIA P160 L. paracasei INIA P773 L. rhamnosus INIA P224 L. salivarius INIA P235 L. rhamnosus INIA P334 L. rhamnosus INIA P374 L. paracasei INIA P393 L. paracasei INIA P461 L. rhamnosus INIA P535 L. rhamnosus INIA P540 L. rhamnosus INIA P556 L. reuteri INIA P568 L. reuteri INIA P574 L. reuteri INIA P580 L. reuteri INIA P581 L. reuteri INIA P582 L. rhamnosus INIA P603 L. paracasei INIA P651 L. paracasei INIA P655 L. paracasei INIA P708
Figure 2. Metabolic pathway of equol.
belong to the INIA culture collection, they were selected from INIA collection for its biotechnological and probiotic properties (Rodrı´guez et al., 2003, 2012). Lactobacillus, Enterococcus, and Bifidobacterium strains were isolated from mother milk and infant feces the healthy infants, with the exception of Lactobacillus reuteri strains which were isolated from pig feces.
Daidzein Genistein Glycitein DHD +
+
+
+ +
-glucosidase activity
+
+ + + +
+
+
+
+
+
+ + +
+
+ + +
+ + + + +
Lactococcus strains were isolated from dairy products as raw milk or cheese. Lactobacillus and Enterococcus were routinely cultivated under anaerobic conditions at 37 C in MRS broth (Oxoid, Ltd. Basingstoke, Hampshire, England). Lactococcus were grown at 30 C in M17 broth (BD, Le Pont de Claix, France) supplemented with glucose (5 g/L) (Sigma Aldrich, Poole, Dorset, UK). Bifidobacterium were cultivated under strict anaerobic conditions at 37 C in RCM broth (BD). Slackia isoflavoniconvertens DSM22006, used as equol-positive control, was grown in Wilkins-Chalgren anaerobe broth (Oxoid) at 37 C under strict anaerobic conditions.
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Table 2. Production of daidzein, genistein, glycitein, dihydrodaidzein (DHD), and -glucosidase activity by Enterococcus strains with biotechnological interest.
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Strains E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E.
Daidzein Genistein Glycitein DHD
faecium INIA P1 faecalis INIA P8 faecalis INIA P14 faecalis INIA P23 faecalis INIA P35 faecalis INIA P52 faecalis INIA P56 faecalis INIA P90 faecium INIA P125 faecalis INIA P127 faecalis INIA P236 faecalis INIA P290 faecalis INIA P333 faecalis INIA P419 faecalis INIA P429 faecium INIA P445 faecium INIA P545 faecium INIA P455 faecalis INIA P464 faecalis INIA P474 faecium INIA P552 faecium INIA P553 faecalis INIA P562
+
+
-glucosidase activity
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
+
+
+ +
+
+ +
+
+ +
Table 3. Production of daidzein, genistein, glycitein, dihydrodaidzein (DHD), and -glucosidase activity by Lactococcus strains with biotechnological interest. Strains L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis L. lactis
Daidzein Genistein Glycitein DHD cremoris ZA24 diacetylactis ZA37 diacetylactis ZA57 diacetylactis ZA86 diacetylactis SJ46 lactis ESI718 lactis TAB26 lactis TAB75 cremoris BO2 diacetylactis BO14 cremoris BO68 diacetylactis BO112 diacetylactis BO128 INIA13 INIA14 INIA15 lactis INIA415 lactis INIA437 cremoris INIA450 lactis ESI153 lactis ESI240 lactis ESI277 lactis ESI515
-glucosidase activity
+
+ + +
+ +
+ + +
+ + +
+ + + + + +
+ + +
+
+
+
+ + + +
+ +
+
Metabolism assay of soybean extracts by LAB and bifidobacteria LAB and bifidobacteria strains were inoculated in 10 mL of BHI medium (Biolife, Milan, Italy) containing 0.5 g/L of l-cysteine supplemented with the soybean extract (4 g/L) (SoyLifes EXTRA; Frutarom Netherlands BV, Veenendaal, the Netherlands), in DMSO, and the pH was adjusted to 7.4 with NaOH. The amount of DMSO present in the media did not affect bacterial growth.
Table 4. Production of daidzein, genistein, glycitein, dihydrodaidzein (DHD), and -glucosidase activity by bifidobacteria strains with biotechnological interest. Strains B. adolescentis INIA P879 B. pseudolongum INIA P2 B. infantis INIA P737 B. breve INIA P712 B. adolescentis INIA P784 B. animalis INIA P797 B. infantis INIA P718 B. longum INIA P911 B. adolescentis INIA P916 B. pseudolongum INIA P965 B. pseudolongum INIA P120 B. longum INIA P678 B. longum INIA P132 B. bifidum INIA P685 B. breve INIA P730 Bifidobacterium sp. INIA P819 B. dentium INIA P882 B. infantis INIA P731 B. pseudocatenulatum INIA P885 B. catenulatum INIA P836 B. catenulatum INIA P926 Bifidobacterium sp. INIA P996 B. animalis INIA P900
Daidzein Genistein Glycitein DHD + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + +
+ + + + +
+ + + + + + + + +
-glucosidase activity + + + + + + + + + + + + + + + + + + + + + + +
The inoculated broths supplemented with the polyphenols were incubated in sealed jars (Oxoid) under anaerobic conditions using AnaeroGen sachets (Oxoid) at 37 C for 7 d. Non-inoculated BHI with soybean extracts was used as control. Extraction of isoflavones After incubations of the inoculated broths, isoflavones were extracted twice with 2 mL of diethyl ether and twice with 2 mL of ethyl acetate according to Gaya et al. (2016). The solvents were evaporated at room temperature under N2 stream and the residue was dissolved in 300 mL methanol/water (50:50, v/v). Filtering through a 0.22 mm cellulose acetate filter (Millipore, Madrid, Spain) was performed before transferring the extracts to HPLC vials and storing them at 20 C. HPLC-PAD analysis Analysis was carried out on a HPLC-PAD Beckman System Gold (Beckman Coulter Inc., Fullerton, CA), comprising an autosampler module 508, binary pump module 126, diode array detector module 168, and 32 Karat Software chromatography manager. Separation of phenolic compounds was achieved on a reverse phase Nova-Pak C18 column (300 3.9 mm, 4 mm) (Waters, Barcelona, Spain). The analytical conditions were based on those described by Gaya et al. (2016). A gradient consisting of solvent A (water/acetic acid, 98:2 v/v) and solvent B
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(water/acetonitrile/acetic acid, 78:20:2 v/v/v) was applied at a flow rate of 1 mL/min from the beginning to 55 min, and 1.2 mL/min from this point to the end. The gradient profile was 0–55 min, 100%–20% A; 55–70 min, 20%– 10% A; 70–80 min, 10%–5% A; 80–110 min, 100% B. Detection was performed by scanning from 210 nm to 400 nm with an acquisition speed of 1 s. A volume of 25 mL was injected. The solvents used, methanol, acetic acid, and acetonitrile were of HPLC grade (LabScan, Gliwice, Poland).
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Identification and quantification of phenolic compounds Chromatographic peaks were identified previously by HPLC-ESI/MS and confirmed by comparison of retention times and characteristics of UV spectra with those of standards (Gaya et al., 2016). The HPLC grade standard compounds daidzein, daidzin, glycitein, equol, genistein, genistin, and glycitin were purchased from LC Laboratories (New Boston Street Woburn, MA). DHD was purchased from Toronto Research Chemicals (TRC) (Toronto, Ontario, Canada). Quantification was made using the external standard calibration curves, with commercial standards (Gaya et al., 2016). Stock solutions of phytoestrogens were prepared in DMSO (Sigma-Aldrich) in a concentration of 10 mg/L. Initial concentrations of daidzein, genistein, and glicitein in control samples were subtracted for calculating the production of these metabolites by the strains analyzed. DHD was not detected in control samples. b-glucosidase activity assays Bacterial cultures were centrifuged at 4000g for 3 min and resuspended in phosphate buffer (0.2 M, pH 6.8). Suspensions were then incubated aerobically for 30 min at 37 C with p-nitrophenyl- -D-glucopyranoside (2.5 mM) (Sigma-Aldrich, St Louis, MO). Release of pnitrophenol was measured with a spectrophotometer (Beckman DU 650 Fullerton, CA) at 405nm before and after incubation (Berg et al., 1978).
Results Isoflavone metabolism was analyzed in 92 strains of bacteria including 23 isolates of each of the genera Lactobacillus, Enterococcus, Lactococcus, and Bifidobacterium. Results of the HPLC analysis of the isoflavone metabolism are listed in Tables 1–4. Fiftythree of the isolates (57.7%) produced at least one of the aglycones studied. Production of daidzein, genistein, and
glycitein from daidzin, genistin, and glycitin in soy extracts was observed in strains of the four genera studied, with the exception of the Lactobacillus genus which did not show any glycitein producer strain. Bifidobacteria were the group showing the greatest ability to metabolize isoflavones since all of Bifidobacterium strains produced at least one compound. Among the other three genera the percentage of strains producing at least one of them was 39.1%, 43.5%, and 47.8% for Lactobacillus, Enterococcus, and Lactococcus respectively. For its part, the isoflavone genistein was the most frequently transformed into its aglycone by all groups of bacteria analyzed and glycitein the least. While production of genistein was observed as the sole compound detected in some isolates, daidzein and glycitein were never detected alone. The analysis of the results reveals that when isoflavone glycosides in soy extracts are converted into their aglycones, genistein is produced. So, production of daidzein or glycitein is always accompanied by the production of genistein in soy extracts. This does not occur in reverse. Then, LAB and bifidobacteria show higher affinity for the transformation of genistin in genistein in soy extracts that the rest of deglycosylations, although the initial concentration of daidzin is much higher. Moreover, the range of production of the isoflavone aglycones was highly variable between strains. Nine Lactobacillus strains produced genistein and only 3 strains produced daidzein; none of the Lactobacillus strains tested produced glycitein (Table 1). Daidzein production from Lactobacillus ranged from 87.67 to 135.64 mg/L and genistein from 38.90 to 209.49 mg/L. The analysis of the metabolism of isoflavones by Enterococcus showed that 9 out of 23 and 10 out of 23 strains were able to produce daidzein and genistein, respectively. Only 4 strains showed glycitein production (Table 2). Daidzein production from Enterococcus ranged from 22.38 to 136.44 mg/L, genistein from 28.25 to 93.53 mg/L and glycitein between 65.98 and 84.58 mg/L. Only one Lactococcus strain was able to produce daidzein, although 11 out of the 23 strains tested produced genistein and two strains glycitein (Table 3). Daidzein production from L. lactis ESI515 was 210.23 mg/L, genistein production by Lactococcus strains ranged from 67.05 to 208.78 mg/L and glycitein from 105.01 to 146.89 mg/L. All the bifidobacteria analyzed produced genistein, and daidzein and glycitein were produced by 78.3% and 60.9% strains, respectively (Table 5). Daidzein production by bifidobacteria ranged from 44.94 to 261.74 mg/L, genistein from 9.47 to 268.99 mg/L, and glycitein from 47.03 to 130.78 mg/L.
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INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION
Initial concentrations of daidzin (665 mg/L), genistin (412 mg/L), and glycitin (214 mg/L) were reduced by the Lactobacillus, Enterococcus, Lactococcus, and Bifidobacterium producing their respective aglycones. Even, some strains completely consumed these initial compounds as L. lactis ESI515. Equol or O-DMA producing strains were not found in the present work, although equol production was observed when S. isoflavoniconvertens DSM22006 was incubated with soy extracts. The precursor metabolite of equol DHD was detected in Lactobacillus rhamnosus INIA P540 (1.09 mg/L) and Enterococcus faecalis INIA P333 (0.87 mg/L). Moreover, E. faecalis INIA P333 was able to produce daidzein, genistein, and glycitein (Figure 3). Fifteen out of 92 isolates (16.3%) were capable of producing glycitein, dadzein, and genistein. Ten Bifidobacterium and four Enterococcus strains produced the three aglycones. Whereas only one of the Lactococcus strain produced the three aglycones. Results from all the bacteria strains studied are summarized in Table 5. -glucosidase enzyme can transform daidzin, genistin, and glycitin into daidzein, genistein, and glycitein,
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respectively, as we observed when LAB and bifidobacteria with -glucosidase activity were incubated in the presence of daidzin, genistin, and glycitin (data not shown). However, results with soy extracts indicate a greater affinity for genistein. -glucosidase activity was confirmed (Tables 1–4) in all the strains able to transform their aglycones by means of the colorimetric method assayed. -glucosidase activity with p-nitrophenyl- -D-glucopyranoside was detected in all soy extracts exhibiting deglycosylation of isoflavones. However, deglycosylation of p-nitrophenyl -D-glucopyranoside to yield p-nitrophenol was observed in strains that did not produce the isoflavone deglycosylation in soy extracts. Fifty-three of the isolates (57.7%) produced at least one of the aglycones studied, whereas sixty four of the isolates (69.6%) showed -glucosidase activity. L. rhamnosus INIA P540 exhibited -glucosidase activity but the strain could not transform daidzein, genistein, or glycitin in its aglycone in soy extracts. Analysis with pure compounds daidzin, genistin, and glycitin showed that this strain was able to produce daidzein, genistein, and glycitein (data not shown).
Table 5. Production daidzein, genistein, glycitein, dihydrodaidzein, and -glucosidase activities by 92 Lactobacillus, Enterococcus, Lactococcus, and Bifidobacterium strains. Lactobacillus Enterococcus Lactococcus Bifidobacterium
Strains
Daidzein
Genistein
Glycitein
DHD
-glucosidase activity
23 23 23 23
3 (13.0%) 9 (39.1%) 1 (4.3%) 18 (78.3%)
9 (39.1%) 10 (43.5%) 11 (47.8%) 23 (100%)
0 4 (17.5%) 2 (8.7%) 14 (60.9%)
1 (4.3%) 1 (4.3%) 0 0
14 (60.9%) 10 (43.5%) 17 (73.9%) 23 (100%)
Figure 3. Identification of isoflavones by HPLC-PAD in soybean extracts control (A). (1, daidzin; 2, glycitin; 3, genistin; 4, dihydrodaidzein; 5, daidzein; 6, glycitein; 7, genistein). In soybean extracts incubated with E. faecalis INIA PRO333 (B).
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Discussion The transformation of glycosylated isoflavones, mainly daidzin, to their respective aglycones by individual bacterial strains or mixed cultures has been reported (Cho et al., 2012; Landete et al., 2014; Otieno et al., 2006; Raimondi et al., 2009; Wei et al., 2007). However, this is the first time a study is made on a large collection of LAB and bifidobacteria. Moreover, this work analyzes the capacity of these strains to metabolize the glycosylated isoflavones daidzin, genistin, and glycitin into their aglycones and secondary metabolites as DHD, O-DMA, and equol. The 92 bacterial strains investigated in the present work were selected from INIA collection for its biotechnological and probiotic properties (Rodrı´guez et al., 2003, 2012). It is noteworthy, as although bacteria with -glucosidase activity are capable of producing the deglycosylation of daidzin, genistin, and glycitin, the results of the tables show how the LAB and bifidobacteria have priority in the production of genistein from genistin, and the daidzein and/or glycitein production do not occur in many cases in soy extracts. One possible explanation is that -glucosidase has more affinity for genistein as already shown (Matsuura & Obata, 1993). The transformation of the glycosylated isoflavones in their aglycone is of great interest for three reasons; increases bioavailability (Selma et al., 2009), increased antioxidant activity (Landete et al., 2014), and facilitates the transformation to equol (Wang et al., 2007). The bioavailability of phenolics is rather low (Manach et al., 2005). This bioavailability can be even lower when the food polyphenols have a large molecular weight, as is the case of complex flavonoid conjugates with several sugars. The microbiota metabolizes these complex polyphenols into smaller molecules which are generally better absorbed in the intestine (Selma et al., 2009). Soybean isoflavones exist as glycosides that are not absorbed intact across the enterocyte of healthy adults because of their higher hydrophilicity and molecular weight (Hur et al., 2000). Their bioavailability requires the conversion of glycosides into aglycones via the action of -glucosidase from bacteria (Izumi et al., 2000). According to our results, a high proportion of LAB and bifidobacteria possess -glucosidase activity and are capable of transforming the glycoside isoflavones into their aglycone, and promote their absorption. Improvement of the antioxidant activity of fermented food products containing glycosylated polyphenols have been described previously. Fermentation of soymilk by L. rhamnosus CRL981 enhanced antioxidant activity and increased isoflavone aglycone content (Marazza et al., 2012). Pyo et al. (2005) demonstrated an increment in
the antioxidant activity of fermented soybean due to the augmented content of aglycones. Landete et al. (2014) demonstrated for the first time the association between the deglycosylation of specific aryl glycosides by L. plantarum 748 with a rise in their antioxidant activity. Increased isoflavone aglycone content in fermented soymilk with L. rhamnosus C6 is likely to improve the biological functionality of soymilk (e.g. antioxidant activity, alleviation of hormonal disorders in postmenopausal women, and so forth) (Hati et al., 2015). Then, LAB and bifidobacteria could be used as functional starter culture to increase the antioxidant activity of plant foods during fermentation. Moreover, De Boever et al. (2001) showed as the interactions between the LAB as L. reuteri and the soygerm powder suggesting the interest that it may be interesting to combine both culturesin fermented milk. The soygerm powder may protect L. reuteri against the bile salt toxicity of the small intestine, whereas the fermentation process releases interesting bioactive isoflavones in the food supplement. Soybean extracts and preparations with LAB and bifidobacteria may be regarded as effective natural and functional dietary food supplements due to their remarkable content of bioactive aglycones and to their significant radical scavenging capacity. The use of probiotic microorganisms in fermented soymilk is beneficial to alter the biological isoflavone aglycone and to improve functional benefits for consumers (Wei et al., 2007). The hydrolysis of glycosides results in metabolites that are potentially more biologically active than the parent compounds (Selma et al., 2009). Furthermore, the glucosides are known to be less bioactive than their respective aglycones (Landete et al., 2015). Daidzein is converted to equol by enzymes of intestinal bacteria in the equol-producer individuals. The clinical effectiveness of soy isoflavones may be a function of the ability to biotransform soy isoflavones to the more potent estrogenic metabolite equol, which may enhance the actions of soy isoflavones, owing to its greater affinity for estrogen receptors, unique antiandrogenic properties, and superior antioxidant activity (Setchell, 2004). Equol is absorbed more efficiently through the colon wall than daidzein (Decroos et al., 2005), appears in plasma after intake of daidzein and remains in plasma for a relatively longer period of time than do genistein and daidzein (Zubik & Meydani, 2003). In this work, we did not find equol-producing strains. Equol production has usually been associated with family Coriobactericeae with the exception of a strain of L. garviae (Uchiyama et al., 2007). More recently, the production of equol from daidzein by B. breve 15700 and B. longum BB536 has been demonstrated
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INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION
(Elghali et al., 2012). However, we did not observed equol production by B. longum BB536 in our laboratory (data not shown). An anaerobic incubation mixture of two bacterial strains Eggerthella spp. Julong 732 and Lactobacillus spp. Niu-O16, that individually transformed DHD to S-equol and daidzein to DHD respectively, produced S-equol from daidzein through DHD. The biotransformation kinetics of daidzein by the mixed culture showed that the production of S-equol from daidzein was significantly enhanced, as compared with the production of S-equol from DHD, by Eggerthella sp. Julong 732 alone (Wang et al., 2007). Then, strains capable of transforming isoflavone glycosides and their aglycones and, essentially, strains capable of producing DHD facilitate the production of equol according to our results So, L. rhamnosus INIA P540 and E. faecalis INIA P333 were able to produce DHD. Moreover, E. faecalis INIA P333 was able to produce daidzein, genistein, and glycitein, being considered as a strain of great interest.
Conclusions LAB and bifidobacteria possessing -glucosidase activity are relevant in the production of compounds with more estrogenic/antiestrogenic and antioxidant activities and bioavailability as daidzein, genistein, glycitein and mainly DHD. Strains with biotechnological and probiotic properties could be further selected for the development of functional fermented soy foods. The ability to metabolize isoflavones can be considered a remarkable characteristic of microorganisms with probiotic potential.
Disclosure statement The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Funding information This work was supported by RM2012-00004-00-00. J.M.L. has a postdoctoral contract with the research program ‘‘Ramo´n y Cajal’’ (MINECO, Spain).
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