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Flavonoid bioconversion in Bifidobacteriumpseudocatenulatum B7003: A potential probioticstrain for functional food development

1756-4646/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jff.2013.12.018

* Corresponding author. Address: Department of Agricultural Sciences, Alma Mater Studiorum-University of Bologna, vialeBologna I-40127, Italy. Tel.: +39 051 2096269; fax: +39 051 2096274.

E-mail address: diana.digioia@unibo.it (D. Di Gioia).

Please cite this article in press as: Di Gioia, D. et al., Flavonoid bioconversion in Bifidobacterium pseudocatenulatum B7003: A potentiastrain for functional food development, Journal of Functional Foods (2014), http://dx.doi.org/10.1016/j.jff.2013.12.018

Diana Di Gioiaa,*, Erwin Strahsburgerb, Ana M. Lopez de Laceyc, Valeria Bregolaa,Ilaria Marottia, Irene Aloisioa, Bruno Biavati a, Giovanni Dinelli a

aDepartment of Agricultural Sciences, Alma Mater Studiorum-University of Bologna, viale Fanin 44, Bologna, ItalybFacultad de Recursos Naturales Renovables, Universidad Arturo Prat, Av. Arturo Prat 2120, Casilla 121, Iquique, ChilecInstituto del Frıo-ICTAN (CSIC), Jose Antonio Novais 10, 28040 Madrid, Spain

A R T I C L E I N F O A B S T R A C T

Article history:

Received 16 July 2013

Received in revised form

17 December 2013

Accepted 19 December 2013

Available online xxxx

Keywords:

Flavonoids

Bifidobacteria

Probiotics

Fermented milks

Soybean

Legumes

Flavonoids are plant derived compounds mainly present as O-glycosides. They are hydro-

lyzed by gut microbial enzymes to their aglycons, which represent the bioavailable and bio-

active form. In this work the capability of a Bifidobacterium strain (Bifidobacterium

pseudocatenulatum B7003) of being used as a probiotic starter culture to obtain fermented

legume milks with an increased concentration of flavonoids in their aglycone form was

studied. B7003 strain can effectively bioconvert glycosylated flavonoids while fermenting

soybean or common bean derived milks. Conversely, a strain used as negative control (Bifi-

dobacterium longum B7254) could grow on the milks but had low activity in flavonoid biocon-

version. B7003 strain possesses basic safety properties, the capability of adhering to gut

epithelial cells and showed resistance to simulated food processing conditions. Thus, B.

pseudocatenulatum B7003 complies well with a new vision of probiotics, which, beside ben-

efits deriving from gut transit, have additional functional properties, such as the high bio-

availability of flavonoids in their aglycone form.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Plants are dietary sources of a large number of phytochemi-

cals. Among them, flavonoids are a large group of secondary

metabolites derived from the phenylpropanoid pathway in

higher plants. They play an important role in plant defense

and nodulation mechanisms (Cooper, 2004; Subramanian,

Stacey, & Yu, 2007). Recently their importance as health-pro-

moting compounds in preventing hormone dependent can-

cers, cardiovascular disease, osteoporosis and menopausal

symptoms have been highlighted by many studies (Ali, Velas-

quez, Hansen, Mohamed, & Bhathena, 2005; Cogolludo et al.,

2007; Cornwell, Cohick, & Raskin, 2004; Di et al., 2008; Ki, Yeo,

& Yhun, 2003; Kottra & Daniel, 2007; Moore et al., 2007; Nagat-

a, 2010; Rossi, Amaretti, Roncaglia, Leonardi, & Raimondi,

2010). These molecules, distributed mainly in leguminous

plants, are naturally occurring estrogen-like compounds

found in seeds and seedlings of soybean (Glycine max L.) (1–

3 mg/g) and other beans such as common bean (Phaseolus vul-

garis L.) (0.2–0.8 mg/g) (Dinelli et al., 2006), contributing to the

intake of bioactive compounds in Asian and Mediterranean

diets, respectively.

Fanin 44,

l probiotic

2 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 4 ) x x x – x x x

Flavonoids are found in plant source mainly as O-glyco-

sides, mainly bound to glucose, but also to other sugars such

as galactose, rhamnose, arabinose and xylose. The O-b-glyco-

sidic bonds of flavonoids, including the isoflavone daidzin

(daidzein-7-O-b-D-glucopyranoside), genistin and glycitin, are

hydrolyzed in the gut mainly by microbial b-glycosidases to

their aglycons, daidzein, genistein and glycitein, respectively

(Day et al., 2000; Hur, Lay, Beger, Freeman, & Rafii, 2000; Schnei-

der & Blaut, 2000; Schneider, Schwiertz, Collins, & Blaut, 1999).

This bacterial transformation is essential for flavonoids’

absorption, bioavailability and functional properties (Jou, Tsai,

Tu, & Wu, 2013; Rossi et al., 2010). Several studies have de-

scribed the b-glucosidase activity of different groups of bacte-

ria, including Bifidobacteria and Lactobacillus strains (Chun

et al., 2007, 2008; Di Cagno et al., 2010; Otieno, Ashton, & Shah,

2006; Raimondi et al., 2009; Rekha & Vijayalakshmi, 2010; Steer,

Johnson, Gee, & Gibson, 2003), which are beneficial microor-

ganisms of the human gastrointestinal tract frequently used

as probiotics in several functional foods (Dore & Corthier,

2010; Granata, Brandi, Borsari, Gasbarri, & Di Gioia, 2012; Sar-

kar, 2013). These bacteria have been found to be active in the

biotransformation of flavonoid glycosides occurring in com-

mon bean seeds and seedlings, as well as in derived foods (Mar-

otti, Bonetti, Biavati, Catizone, & Dinelli, 2007; Otieno et al.,

2006; Raimondi et al., 2009; Rekha & Vijayalakshmi, 2010).

A number of fermented products derived from cow’s milk

contain probiotic bacteria, which are often directly involved

in the fermentation process. Conversely, fermented products

based on different substrates, such as soybean or other le-

gumes, have little applications in western countries although

they are widely diffused in eastern countries (Lee, Lo, & Pan,

2013). Legumes not only have a high protein content (20–

40%), but are also rich in other nutritional compounds, such

as carbohydrates, B-complex vitamins like thiamine, niacin

and minerals such as calcium, magnesium, zinc, iron and

phosphorous (Garg & Dahiya, 2003). Soymilk fermented by lac-

tic acid bacteria has good nutritional and healthy properties

(Marazza, LeBlanc, de Giori, & Garro, 2013; Molina, Medici, Font

de Valdez, & Taranto, 2012). Moreover, it is a good medium for

growing bifidobacteria, because it contains sugars that are fer-

mented by most member of this genus (Scalabrini, Rossi, Spet-

toli, & Matteuzzi, 1998). Conversely, no studies have been

focused on common bean derived milk.

Bifidobacterium pseudocatenulatum B7003 strain possesses

high b-glucosidase activity and the capability of converting

the flavonoids of Phaseulus vulgaris seeds and seedlings, such

as kaempferol 3-O-glucoside, daidzin, genistin and glycitin,

into their aglycone forms without further metabolization (Mar-

otti et al., 2007). Considering that B. pseudocatenulatum is one of

the most abundant bacterial species in the human intestinal

tract and that probiotic research aims at developing functional

food products able to modify the gut microbiota to a potentially

healthier one (Gaggia, Di Gioia, Baffoni, & Biavati, 2011; Sarkar,

2013), B7003 strain may affect flavonoid-based products

enriching their content in bioavailable aglycone forms and, at

the same time, beneficially affecting the gut.

The main objective of the present contribution was to

investigate potential applications of B. pseudocatenulatum

Please cite this article in press as: Di Gioia, D. et al., Flavonoid bioconversiostrain for functional food development, Journal of Functional Foods (2014

B7003 strain as a probiotic starter culture to obtain fermented

legume milks with an increased concentration of bioavailable

flavonoids in their aglycone form. Probiotic characteristics

were studied in term of survival in simulated gastrointestinal

(GI) tract conditions and presence of antibiotic resistances.

Moreover, survival in food processing conditions was

evaluated.

2. Methods and materials

2.1. Chemicals

Reagents for culture media preparation and man rogosa sharpe

(MRS) broth were from Oxoid (Basingstoke, UK); anaerobic con-

ditions were obtained with an anaerobic atmosphere genera-

tion system (Anaerocult A, Merck, Darmstadt, Germany). All

solvents used for high-performance liquid chromatography

(HPLC) separation were HPLC grade from Carlo Erba, Milan.

For b-glucosidase activity the substrate (p-nitrophenyl-b-

glucopiranoside) and the product (p-nitrophenol) were from

Sigma–Aldrich, Milan (Italy). Authentic standards of kaempfer-

ol 3-O-glucoside and kaempferol were purchased from Indo-

fine Co. (Hillsborough, NJ). Isoflavone glycosides (daidzin,

genistin and glycitin) and related aglycones (daidzein, geni-

stein and glycitein) were from Extrasynthese (Genay, France).

2.2. Strain and culture conditions

B. pseudocatelunatum B7003 strain used in the study was cho-

sen considering its high capability of bioconverting glycosyl-

ated flavonoids into their aglycone forms in extracts

obtained from common bean seed and seedlings (Marotti

et al., 2007). Bifidobacterium longum B7254, which was found

not to possess detectable b-glucosidase activity, was selected

as the negative control. Strains were activated by two succes-

sive transfers in trypticase phytone yeast extract (TPY) med-

ium (Santini et al., 2010). An inoculum of 5% (v/v) was used

for strain cultivation, and incubation was carried out anaero-

bically at 37 �C for 24 h (Santini et al., 2010). Viable cell counts

were determined in triplicate by using the standard plate

counts on TPY agar and were expressed as colony-forming

units per milliliter of culture (CFU/mL).

2.3. Preparation and fermentation of soy and bean derivedmilk and fermentation of commercial soymilk

P. vulgaris L. seeds of the cultivar ‘‘Verdone’’ were purchased

from the seed producer Melandri Gaudenzio, Ravenna, Italy.

The seeds of P. vulgaris L. landrace ‘‘Zolfino del Pratomagno’’

were directly obtained from local farmers (Reggello, Arezzo,

Italy). One soybean (Glycine max L.) cultivar ‘‘Aires’’ was also

included in the study. Whole seeds of soybean and common

bean were washed and soaked in distilled water. After decant-

ing the water, they were crumbled in a blender using distilled

water (1:6% w/v). The resultant slurry was filtered with seven

layers of cheese cloth to obtain soymilk or beanmilk. In addi-

tion, a commercial soymilk (Valsoia) was used in fermenta-

tion studies.

n in Bifidobacterium pseudocatenulatum B7003: A potential probiotic), http://dx.doi.org/10.1016/j.jff.2013.12.018

J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 4 ) x x x – x x x 3

Fermentation was performed in tubes each containing

5.0 mL of medium. Three tubes for each sampling time were

prepared for each substrate used. The fermentation medium

was composed of: 4.45 mL of soymilk or beanmilk, 0.05 mL

of 50 mg/mL L-cystein and 0.5 mL of inoculum. The inoculum

was prepared with an overnight TPY culture, centrifuged and

suspended in PBS buffer in order to obtain an A600 of 0.4. Not

inoculated tubes were used as controls. Sampling times were

soon after inoculum (time 0), 9, 24, 48 and 72 h of incubation.

At each sampling time, 0.1 mL of the tube content was used

for cell count evaluation via standard plate counts on TPY

agar plates and the same amount was used for pH measure-

ment. The remaining medium was extracted to evaluate the

content of flavonoids.

2.4. Extraction and HPLC analyses

Sep-Pak C18 cartridges (Agilent technologies, Milan, Italy)

were activated by 3 mL of ethyl acetate, 3 mL of methanol

and 3 mL of water. Each supernatant obtained from bacterial

culture fermentation was added to the column, and eluted

with 2 mL of ethyl acetate to collect the metabolites for HPLC

analysis. Each extract was vacuum-dried, dissolved in 0.5 mL

of 80% methanol solutions, and stored at �20 �C until analy-

sis. Flavonoids were analyzed by HPLC-DAD (Beckman) fol-

lowing the protocol of Marotti et al. (2007).

2.5. Survival in simulated GI tract conditions andadhesion to intestinal epithelial cell lines

The survival assays were performed with an overnight culture

of strain B7003 in TPY medium (A600 of about 0.9). Cells were

centrifuged, washed in PBS and suspended in the same buffer

to an A600 of 0.7 (corresponding to about 8.5 Log CFU/mL).

Resistance to acidity was evaluated by using PBS at different

pH values (2.5, 3.5 and 4.5); resistance to bile salts was exam-

ined preparing a PBS solution at pH 7.0 with 1 and 2% bile

salts (Ox-bile LP0055, Oxoid). The suspensions were incubated

anerobically at 37 �C and enumerated after 0, 0.5, 1 and 4 h. A

cell suspension at pH 4.5 was also maintained for 1 week at

4 �C in anaerobic conditions and cell survival was evaluated.

Adhesion to small intestinal human epithelial H4 cell

monolayers was performed as described in Aloisio et al.

(2012). Briefly, B. pseudocatenulatum B7003 was applied to H4

cell monolayers (prepared according to Aloisio et al. (2012))

at a concentration of 9.4 Log (CFU/m2). Results of attached

bacteria cells were expressed as percentage of adherent bac-

terial cells compared to initial inoculum and results were

compared with those obtained with the well known probiotics

strain LGG (Lactobacillus rhamnosus GG).

2.6. Survival to simulated food processing conditions

The strain resistance to starvation was examinated in PBS pH

7; resistance to thermal stresses was studied in PBS pH 7

pre-heated at 50 or 55 �C; resistance to osmotic pressure

was studied in PBS pH 7 with 2 and 4% NaCl. Cell suspensions,

prepared as described above, were incubated anerobically at

37 �C or at the desired temperature for thermal stresses and

enumerated after 0.5, 1 and 4 h of exposure. Viable cells were

Please cite this article in press as: Di Gioia, D. et al., Flavonoid bioconversistrain for functional food development, Journal of Functional Foods (2014

determined analyzing 100 lL of each culture with the micro-

dot methods (Strahsburger, Baeza, Monasterio, & Lagos,

2005) on TPY agar plates and cultured anaerobically at 37 �C.

2.7. Antibiotic resistance characterization

The following antibiotic agents, all suggested in the European

Food Safety Authority (EFSA) guidelines (EFSA, 2008), were

used: ampicillin, cefuroxime and vancomycin, as cell wall syn-

thesis inhibitor; chloramphenicol, erythromycin, kanamycin,

streptomycin and tetracycline as protein synthesis inhibitors.

All antibiotics were obtained from Sigma (St. Louis, MO, USA).

The procedure used for minimal inhibitory concentration

(MIC) has been described in Aloisio et al. (2012). Briefly, TPY

broth containing antibiotics at different concentrations (0.5–

500 lg/mL) was prepared and introduced in the wells of a mi-

cro-well plate. The inoculum for the wells was adjusted to a

concentration of 5 · 105 CFU/mL. The experiments were repli-

cated at least three times. The characterization of sensitivity/

resistance was done according to the guidelines and break-

points defined by EFSA (EFSA, 2008).

The presence of known antibiotic resistance genes was

checked via PCR using specific primers: aph (300)-I, aph (300)-

II, aph (300)-III coding for kanamycin and neomicine resistance

(Ouoba, Lei, & Jensen, 2008), aadA, aadE, ermA, streptomycin

and erythromycin-resistance genes (Ouoba et al., 2008), tet(M),

tet(O), tet(W) coding for tetracycline resistance (Masco, Van

Hoorde, De Brandt, Swings, & Huys, 2006) and blaCTX-M-g1,

blaCTX-M-g2, ß-lactam resistance (Van Hoek & Aarts, 2008).

The amplification conditions are from Ouoba et al. (2008).

The annealing temperature varied in the range 45–64 �C,

depending on the primer. Lactobacillus casei L9 was used as po-

sitive control for aph(300)-III, aadA, aadE genes whereas Bifido-

bacterium adolescentis DSM 20087 was the positive control for

tet(W) gene. PCR products were separated by electrophoresis

on 1.5% agarose gel.

Plasmid content in the B7003 strain was checked as de-

scribed in Aloisio et al. (2012).

2.8. Experimental design and statistical analysis

For the different trials, the adopted experimental scheme was

a fully randomized design. All the tests were performed in

triplicate. Data on glycoside flavonoid conversion, survival to

simulated gastrointestinal tract and industrial processing con-

ditions, antibiotic susceptibility were subjected to one-way

analysis of variance (ANOVA) by using the GLM procedure of

the SAS statistical package. Means were subjected to Fisher’s

test (SAS 1988). When treatments were significant according

to Fisher’s test, corresponding means were differentiated by

the SNK multiple range test at the 0.05 level of probability.

3. Results and discussion

3.1. Fermentation of soymilks and bean derived milks

The capability of B. pseudocatenulatum B7003 and of B. longum

B7254 strains of growing on a commercial soy milk, a soymilk

prepared with the cultivar Aires and two bean derived milks

on in Bifidobacterium pseudocatenulatum B7003: A potential probiotic), http://dx.doi.org/10.1016/j.jff.2013.12.018

4 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 4 ) x x x – x x x

was evaluated. Previous studies have shown that the capabil-

ity of Bifidobacterium spp. to grow on soymilks is not a specie-

specific feature and depends on the strain (Tsangalis, Ashton,

McGill, & Shah, 2002). B. pseudocatenulatum strains have never

been assayed for this purpose. Viable counts of the two Bifido-

bacterium strains in the commercial and Aires-derived soy-

milk is shown in Fig. 1 in comparison with growth on TPY

broth. B7003 strain could grow effectively in soymilks: expo-

nential growth on the commercial soymilk was observed over

the 1st and 9th h of incubation, reaching a 3 Log increase in

this time. Growth on soya Aires milk was of less extent (a 2

Log increase was observed over the 1st and 24th h of incuba-

tion) but, however, it was comparable to that on TPY medium.

Subsequently, viable counts on all soymilks rapidly de-

creased. The fast cell death may indicate a diminishing con-

centration of fermentable substrates, as already evidenced

in the works of Tsangalis et al. (2002). pH was 7.5 and 6.8 at

the beginning of the fermentation in the commercial soymilk

and in the milk from soya cultivar Aires, respectively, and

reached a value of 4.5 in both samples after 24 h in the com-

mercial milk and between 24 and 48 h for the soya Aires milk.

This drop in pH, determined by acid metabolites production

by Bifidobacterium strains, caused soymilk coagulation as incu-

bation proceeded. Results reported in the following sections

of this contribution showed a good capability of the B7003

strain of surviving at pH 4.5; therefore, on the whole, the data

obtained indicated 12 and 24 h as the optimal fermentation

times with no pH control for the commercial soymilk and

the milk from soya cultivar Aires, respectively. B. longum

B7254 strain evidenced a capability of growing on both soy-

milks similar to the B7003 strain (Fig. 1B), but the start of

growth on soymilks was slower.

Fig. 1 – Viable counts of B. pseudocatenulatum B7003 (A) and

B. longum B7254 (B) in the commercial (m) and Aires-derived

(h) soymilks in comparison with growth on TPY broth (s).

Please cite this article in press as: Di Gioia, D. et al., Flavonoid bioconversiostrain for functional food development, Journal of Functional Foods (2014

In addition, a new legume source for obtaining fermented

milks was assayed. B7003 strain showed a very good capabil-

ity of growing on the milks obtained from the two P. vulgaris

cultivars used (Fig. 2A). In the initial 24 h of incubation,

growth was even faster and of great extent with respect to

the TPY medium. As already observed for the soya Aires milk,

cell death was more rapid with respect to the TPY medium as

the incubation proceeded and a pH drop was observed. pH

reached the value of 4.0 both in the zolfino and in the verdone

milk between the 24th and 48th h of incubation. On the con-

trary, the B7254 strain had a limited growth on the two bean

milks (only 1 Log of cell increase after 24 h of incubation),

whereas growth on TPY was comparable to that of B7003

strain, Fig. 2B).

3.2. Glycoside flavonoid conversion in soymilks and beanderived milks

It is well known that flavonoids in fermented foods are more

available to humans than those present in non-fermented

products (Di Cagno et al., 2010). The ability of investigated bif-

idobacteria strains of converting isoflavone and kaempferol

glucosides into bioactive aglycones during soyamilk and

beanmilk fermentation was investigated (Table 1). HPLC anal-

yses showed that daizdin, genistin and glycitin, present in the

commercial soymilk and in the Aires derived milk, were con-

verted to the corresponding aglycones (didzein, genistein and

glycitein) with different rate constants (Table 2). According to

Eisen, Ungar, and Shimoni (2003) and Otieno et al. (2006), a

first order kinetics has been assumed to determine degrada-

tion constants for the individual isoflavone and kaempferol

glycosides. Plotting ln [C]/[C0] (where C is the concentration

Fig. 2 – Viable counts of B. pseudocatenulatum B7003 (A) and

B. longum B7254 (B) in the verdone (m) and zolfino (h)

beanmilks in comparison with growth on TPY broth (s).

n in Bifidobacterium pseudocatenulatum B7003: A potential probiotic), http://dx.doi.org/10.1016/j.jff.2013.12.018

Table 1 – Glycoside flavonoids and their aglyconic form in soymilks (commercial and Aires home-made soymilk) andbeanmilks (Verdone and Zolfino home-made beanmilks).

Source Glycoside flavonoids Aglycones

Valsoia (commercial soymilk) Daidzin Didzein

Genistin Genistein

Glycitin Glycitein

Aires (home-made soymilk) Daidzin Didzein

Daidzein malonyl

Genistin Genistein

Genistein malonyl

Glycitin Glycitein

Verdone (home-made beanmilk) Kaempferol 3-O-glucoside Kaempferol

Zolfino (home-made beanmilk) Kaempferol 3-O-glucoside Kaempferol

Table 2 – Flavonoids conversion in their aglyconic form in soymilks and bean derived milks according to first orderkinetics. C0 = initial concentration of the glycoside flavonoid expressed as lmol of flavonoid per gram of seed; K = rateconstant; R2 = coefficient of determination; P = statistical significance level (different letters indicate mean values differentat P < 0.05); CT50 = time (h) required for the conversion of 50% of the initial glycoside flavonoid into the respectiveaglycones and SD = Standard deviation of the CT50.

Glycoside flavonoid Source Strain C0 (lmol/g) K (h�1) R2 P CT50 (h) SD

Daidzin Valsoia B7003 6.518 0.207 0.998 c 3.4 0.1

Genistin Valsoia B7003 6.860 0.391 0.998 a 1.8 0.0

Glycitin Valsoia B7003 1.113 0.339 0.999 b 2.0 0.1

Daidzin Aires B7003 1.751 0.055 0.906 h 12.7 0.1

Genistin Aires B7003 1.641 0.149 0.821 e 4.6 0.0

Glycitin Aires B7003 0.242 0.176 0.990 d 4.0 0.4

Daidzin Valsoia B7254 6.518 0.036 0.892 ij 19.5 1.1

Genistin Valsoia B7254 6.860 0.082 0.851 g 8.4 0.5

Glycitin Valsoia B7254 1.113 0.023 0.949 jk 30.4 3.2

Daidzin Aires B7254 1.751 0.010 0.972 kl 68.7 2.9

Genistin Aires B7254 1.641 0.030 0.828 j 22.9 0.4

Glycitin Aires B7254 0.242 0.001 0.990 l 514.1 26.9

Daidzein malonyl Aires B7003 1.731 0.125 0.856 f 5.5 0.4

Genistein malonyl Aires B7003 1.549 0.149 0.911 e 4.7 0.6

Daidzein malonyl Aires B7254 1.731 0.001 0.828 l 1359.6 37.7

Genistein malonyl Aires B7254 1.549 0.000 0.818 l 1401.6 60.1

K 3-O-glucoside Verdone B7003 2.672 0.044 0.823 hi 15.6 0.8

K 3-O-glucoside Zolfino B7003 1.164 0.032 0.845 ij 21.5 0.1

K 3-O-glucoside Verdone B7254 2.672 0.003 0.814 l 252.1 10.5

K 3-O-glucoside Zolfino B7254 1.164 0.005 0.900 l 149.1 9.3

J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 4 ) x x x – x x x 5

of the glycoside flavonoid at a certain time, and C0 is the ini-

tial concentration of the glycoside flavonoid) against time, in

accordance with a first order kinetic straight lines have been

obtained, as evidenced by the observed coefficients of deter-

mination (R2), ranging between 0.814 and 0.998 (Table 2).

With regards to isoflavones, the B7003 strain exhibited a

significantly higher b-glucosidase activity with respect to

B7254 strain (Table 2). Considering the soy milk source and

the type of glycoside isoflavone, the mean rate constant was

0.219 ± 0.128 and 0.030 ± 0.03 h�1 for the B7003 and B7254

strain, respectively. For the B7003 strain the 50% conversion

time (CT50) of different glycoside isoflavones into the respec-

tive aglycones ranged between 1.8 and 12.7 h, while for the

B7254 the same conversion required a time ranging between

Please cite this article in press as: Di Gioia, D. et al., Flavonoid bioconversistrain for functional food development, Journal of Functional Foods (2014

8.4 and 514.1 h. The b-glucosidase activity of both bifidobacte-

ria strains was strongly dependent on the preparation of soy

milk: a relatively slow conversion was observed for the Aires

home-made soy milk (total mean rate constant equal to

0.07 ± 0.07 h�1) with respect to commercial Valsoia prepara-

tion (total mean rate constant equal to 0.180 ± 0.158 h�1).

The observed matrix effect is presumably associated with

the different purity and contaminant level between home-

made and commercial soybean milk, affecting the enzymatic

activities of bifidobacteria strains. B7003 and B7254 strain

exhibited a similar affinity in converting different glycoside

isoflavones: the highest conversion was observed for genistin

(mean rate constants equal to 0.237 ± 0.212 and 0.09 ± 0.08 h�1

for B7002 and B7254 strain, respectively), while the lowest

on in Bifidobacterium pseudocatenulatum B7003: A potential probiotic), http://dx.doi.org/10.1016/j.jff.2013.12.018

6 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 4 ) x x x – x x x

b-glucosidase activity is referred to daidzin (mean rate con-

stant equal to 0.121 ± 0121 and 0.032 ± 0.031 h�1 for B7003

and B7254 strain, respectively). Glycitin showed an intermedi-

ate conversion rate with values more similar to those ob-

served for genistin (mean rate constants equal to

0.181 ± 0.222 and 0.088 ± 0.123 h�1 for B7003 and B7254 strain,

respectively).

Isoflavone malonyl forms (i.e., daidzein malonyl and geni-

stein malonyl) have been exclusively found in the Aires

home-made soy milk. The affinity of B7254 strain for malonyl

isoflavones was particularly low, as evidenced by the respec-

tive mean rate constants and CT50 values, ranging between

1359 and 1401 h. In contrast, B7003 strain has converted mal-

onyl forms with the same extent observed for the glycoside

isoflavones (Table 2).

The two bifidobacteria strains showed significant differ-

ences in converting kaempferol 3-O-glucoside. The B7003

strain converted 50% of this flavonoid into its respective agly-

cone form in less than one hour (total mean rate equal to

0.038 ± 0.008 h�1), while for B7254 the same conversion re-

quired a time ranging from 149 to 252 h (total mean rate

0.004 ± 0.001 h�1). The b-glucosidase activity of both bifido-

bacteria strains was independent of the source of kaempferol

3-O-glucoside (i.e., bean milk from Verdone or Zolfino bean).

3.3. Characterization of B7003 strain as a potentialprobiotic

3.3.1. Survival in simulated gastro-intestinal tract conditionsand adhesion to epithelial cellsThe ability of a potential probiotic strain to survive to the

transit in the gastrointestinal tract of the consumer (low pH

of the stomach and bile in the upper small intestine) and ad-

here to the gut epithelium is extremely important (Sheehan,

Sleator, Hill, & Fitzgerald, 2007). Cell suspension of B. pseudo-

catenulatum B7003 with 1% and 2% bile salts conserved the ini-

tial population for 2 h, decaying only 1 log after 4 h exposure

at both concentrations assayed (Table 3). Gastric pH (i.e., low-

er than 3) strongly affected the viability of B7003 strain, which

is a frequent feature in Bifidobacterium spp. strains (Dunne

et al., 1999; Santini et al., 2010); however, it is known that a

proper choice of the food carrier, such as a food matrix, for

the probiotic strain may enhance its survival in the stomach

due to the local increase of pH (Clegg & Shafat, 2010). There-

fore, the capability of the B7003 strain of surviving to slightly

higher pH with respect to the stomach one such as 3.5 and 4.5

is extremely important for its survival during the stomach

transit within a food matrix. In addition, as already pointed

Table 3 – Survival of B. pseudocatenulatum B7003 to simulatewith respect to time 0).

Time (hours) Survival percentage

pH 4.5 pH 3.5

0.5 100 100

1 97.2 95.8

4 95.8 62.5

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out, survival at pH 4.5 is important considering that several

fermented milks can have an acidic pH at the end of the fer-

mentation process (Wang, Yu, & Chou, 2002); the survival

after 1 and 4 h exposure to these pH values is almost com-

plete (Table 3). A cell suspension of B7003 was also main-

tained at pH 4.5 for 1 week at 4 �C in anaerobic condition

and the survival was found to be 80% of the initial cell

amount, thus indicating a high survival in acidic pH at storage

conditions (data not shown).

The B7003 strain also showed a good ability to in vitro ad-

here to epithelial H4 cells, which was even higher with re-

spect to the LGG strain: 9.1 and 8.8 Log(CFU/m2) cells were

recovered after the adhesion test in the B7003 and LGG as-

says, respectively, out of the 9.4 Log(CFU/m2) cells initially ap-

plied. The ability of a potential probiotics strain to adhere to

the intestinal epithelium is a very important features as it al-

lows to persist in the gut preventing its elimination by peri-

stalsis and the adhesion of pathogenic bacteria.

3.3.2. Survival to simulated food processing conditionsThe potential of the B7003 strain of surviving to simulated

food processing conditions (starvation, reduced water activity

and thermal stresses) were also assayed and results are pre-

sented in Table 4. The strain was able to resist well to starva-

tion stress, maintaining almost all the initial population after

24 h of suspension in PBS, it was viable at 50 and 55 �C for 1 h

after the application of the thermal stress, decaying only 2 log

of the initial cell population. After 4 h of incubation, no cells

survived to both temperature stresses. However, considering

that bifidobacteria are generally sensitive to high temperature

(Schmidt & Zink, 2000), the tolerance of B7003 to 1 h exposure

to such high temperatures is of great significance; it is in fact

known that transient contacts with high temperatures can

occur during microorganism drying and storage processes in

the food industry (Meng, Stanton, Fitzgerald, Daly, & Ross,

2008; Simpson, Stanton, Fitzgerald, & Ross, 2005). The addi-

tion of 2% and 4% NaCl to increase the osmotic pressure only

slightly affected survival after 24 h of incubation.

3.3.3. Haemolysis and antibiotic resistanceStrain B7003 did not show any haemolytic activity on blood

agar plate (data not shown), in agreement with other studies

showing that haemolysis is rarely present in bifidobacteria

(Meile, Le Blay, & Thierry, 2008). Resistance or sensitivity to

the selected antibiotics and the relative MIC values obtained

are shown in Table 5; according to the FEEDAP 2008 guidelines

(EFSA, 2008), the strain was sensitive to Erythromycin, Vanco-

mycin and Chloramphenicol. It also showed a low MIC value

d GI tract conditions (expressed as percentage of survival

pH 2.5 Bile salts 1% Bile salts 2%

30 100 100

0 96.5 97.5

0 81.2 75.9

n in Bifidobacterium pseudocatenulatum B7003: A potential probiotic), http://dx.doi.org/10.1016/j.jff.2013.12.018

Table 4 – Survival of B. pseudocatenulatum B7003 to simulated industrial processing conditions (expressed as percentageof survival with respect to time 0).

Time (hours) Survival percentage

Starvation Thermal stress (50 �C) Thermal stress (55 �C) NaCl 2% NaCl 4%

0.5 100 65.3 65.5 100 100

1 100 65.3 63.1 100 100

4 100 0 0 100 100

24 95.3 –* –* 91.8 88.2

* – Not determined.

Table 5 – MIC of various antibiotics of B. pseudocatenul-atum B7003 strain. Strain are characterized as sensitiveor resistance according to the breakpoints defined by theEFSA (2008) guidelines.

Antibiotics MIC (mg/ml) Susceptibility

Ampicillin >500 Resistant

Kanamycin >500 Resistant

Streptomycin >500 Resistant

Tetracycline 16 Resistant

Erythromycin 1 Sensitive

Vancomycin 1 Sensitive

Chloranmphenicol 0.5 Sensitive

Cefuroxime 0.5 Sensitive*

* The breakpoint of cefuroxime is not defined in the EFSA guidelines,

the strain has been considered sensitive because of the low MIC.

J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 4 ) x x x – x x x 7

to Cefuroxime, for which the antibiotic resistance breakpoint

is not available in the EFSA guidelines. On the contrary, the

strain was resistant to tetracycline, kanamycin, streptomycin

and ampicillin. Although tetracycline, kanamycin and strep-

tomycin resistances are widely diffused among bifidobacteria

(Ammor, Florez, Alvarez-Martın, Margolles, & Mayo, 2008;

D’Aimmo et al., 2007; Kiwaki & Sato, 2009), the genetic mech-

anism of antibiotic resistance in bifidobacteria has not been

completely elucidated and only some genes coding for tetra-

cycline resistance, such as tet(W) and tet(M), have been widely

characterized in Bifidobacterium spp. strains (Aires, Doucet-

Populaire, & Butel, 2007; Ammor et al., 2008; Meile et al.,

2008). Screening for resistance genes towards antibiotics to

which B7003 was resistant was performed with the use of

specific primers (detailed in Section 2). The only positive reac-

tion was found with tet(W), which is the most widely distrib-

uted tetracycline resistance gene among Bifidobacterium spp.

(Aires et al., 2007), whereas no amplification was obtained

with the primers specific for aph(300)III, aadE, blaCTX-M1 and

blaCTX-M2. The aph(300)III gene, coding for kanamycin resis-

tance, has been evidenced in Lactobacillus strains, but never

among bifidobacteria (Ouoba, Lei, & Jensen, 2008), whereas

the streptomycin resistance gene aadE has been evidenced

in a B. longum strain (Ouoba et al., 2008). In addition, the

search for b-lactam antibiotic resistance with the blaCTX-M1

and blaCTX-M2 specific primers, which were used to detect

this resistance in Enterobacteriaceae (Van Hoek & Aarts,

2008), had no success, although the blaCTX-M-g1 is quite dif-

fused among bifidobacteria (Aloisio et al., 2012). No plasmids

were detected in the B7003 strain and this represent an

Please cite this article in press as: Di Gioia, D. et al., Flavonoid bioconversistrain for functional food development, Journal of Functional Foods (2014

important feature considering that transmissible antibiotic

resistances are in the majority of strains encoded by plasmid

DNA. Therefore, the strain considered possess multiple resis-

tances to antibiotics, but they are all chromosomally encoded

and, therefore, their spread to other bacteria can be consid-

ered a rare event.

4. Conclusion

B. pseudocatenulatum B7003 strain has a great potential for

being used as a starter culture for the fermentation of legume

derived milks with increased functional properties. This

strain is capable of effectively bioconverting glycosylated

flavonoids to the aglycone forms while fermenting soya and

bean derived milks, thus improving their bioavailability. On

the contrary, B. longum B7254 strain can grow on the milks

but has a low activity in glycosylated flavonoid bioconversion.

Therefore, the choice of the strain for flavonoid bioconversion

is fundamental. In addition to the bioconversion activity, the

B7003 strain has interesting probiotic characteristics, as it

possesses basic safety properties, the capability of adhering

to gut epithelial cells and a high resistance in simulated food

processing conditions. In conclusion, the B. pseudocatenulatum

B7003 strain complies well with a new vision of probiotic

strains, which, beside traditional benefits deriving from their

transit in the gut, may have additional functional properties

for the consumers, such as the high availability of flavonoids

in their aglycone form. These properties can be exploited in

the development of functional foods containing probiotic

strains. Last but not least, this work has shown the potential

of other legumes different from soya beans of becoming inter-

esting ingredients for functional fermented products.

Acknowledgments

The study was funded by the University of Bologna, RFO Pro-

gram number J61J10000790001. Erwin Strahsburger received a

scholarship within the Erasmus Mundus Program. Ana M. Lo-

pez de Lacey received a fellowship within the JAE-CSIC pre-

doctoral Programme.

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