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Soybean-based functional food with vitamin B12-producinglactic acid bacteria

Veronica Molinaa, Marta Medicia, Graciela Font de Valdeza,b, Marıa Pıa Tarantoa,*

aCentro de Referencia para Lactobacilos (CERELA) – CONICET, Chacabuco 145, T4000LC San Miguel de Tucuman, ArgentinabCatedra de Microbiologıa Superior, Facultad de Bioquımica, Quımica y Farmacia, Universidad Nacional de Tucuman,

San Miguel de Tucuman, Argentina

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

Article history:

Received 16 January 2012

Received in revised form

23 May 2012

Accepted 24 May 2012

Available online 27 June 2012

Keywords:

Functional food

Lactobacillus reuteri

Soy-based beverage

Vitamin B12

1756-4646/$ - see front matter � 2012 Elsevihttp://dx.doi.org/10.1016/j.jff.2012.05.011

* Corresponding author: Tel.: +54 381 431 046E-mail address: ptaranto@cerela.org.ar (M

Consumption of soy products has increased greatly due to their beneficial health effect.

However, soy does not contain vitamin B12 (B12), an important water-soluble vitamin vital

to prevent severe pathologies, some of which are irreversible. In this study a novel soymilk

beverage containing a compound with B12 activity-producer strain (Lactobacillus reuteri CRL

1098) to prevent the pathologies caused by a B12-deficient diet was evaluated using an

experimental murine model. Pregnant females were divided into four groups. Besides the

B12-deficient and the control group, the animals in the remaining two groups received

non-fermented soymilk and soymilk fermented with L. reuteri CRL 1098 from the end of ges-

tation to weaning. At the end of the trials, females and their corresponding offspring were

sacrificed to determine haematological, immunological and histological parameters. The

results showed that the administration of fermented soymilk prevented the development

of all symptoms observed as a consequence of nutritional B12 deficiency both in females

mice and in their respective offspring. The design of soybean-based functional food bio-for-

tified with vitamin using a lactobacilli strain able to produce compounds with B12 activity

constitutes an interesting alternative therapy to prevent vitamin deficiency.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The beneficial effect (probiotic) that some lactic acid bacteria

(LAB) exert on the health of consumers has led to the develop-

ing of certain functional foods. Although the LAB are

commonly associated with dairy products, this group of micro-

organisms also plays a role in other food systems such as sau-

sages and drinks, food conservation, olive ripening and

sourdough bread, among others. The versatility of LAB encour-

ages scientists to search for new applications to obtain novel

products for a continuously growing functional food market.

Besides dairy products, soy-based beverages are an inter-

esting option in nutrition considering their high nutritional

value, the quality of the protein and amino acids and the

er Ltd. All rights reserved

5; fax: +54 381 400 5600..P. Taranto).

low production costs of soybeans. However, this substrate

presents some disadvantages such as low vitamin content,

mainly the water-soluble vitamin B12 (Riaz, 2006) belonging

to the B complex group. This vitamin is involved as a cofactor

in a variety of enzymatic reactions and as a methyl donor in

the synthesis of DNA and red blood cells. It is essential to

maintain the integrity of the insulation sheath (myelin

sheath) that surrounds the nerve cells (Miller, Korem, Almog,

& Galboiz, 2005).

A diet sufficient in vitamin B12 is essential to prevent severe

pathologies, some of which are irreversible. It has been

reported that vitamin B12-deficiency in the diet of pregnant fe-

males causes severe retardation of myelination in the nervous

system and brain atrophy in the infants (Lovblad, Ramelli, &

.

832 J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 8 3 1 – 8 3 6

Remonda, 1997; Pepper & Black, 2011). During infancy, this

deficiency may cause failure to thrive, irritability and delay

of neurological development, convulsions, and even severe

megaloblastic pancytopenia due to a delayed DNA synthesis

and myelination defects (Grattan-Smith, Wilcken, Procopis,

& Wise, 1997; Ramussen, Fernboff, & Scanlon, 2001; Taskesen,

Yaramis, Pirinccioglu, & Ekici, 2011).

Cobalamin is exclusively synthesized by certain bacteria

and archaea. In cattle, sheep, and other ruminants, microor-

ganisms present in the rumen can synthesize cobalamin. Hu-

mans, however, do not have such microflora in their small

intestine and they must absorb the coenzyme from natural

sources such as animal organ meats (especially liver and

kidney), fish, eggs, and pharmaceutical products (Watanabe,

2007).

In a previous paper, we reported the ability of Lactobacillus

reuteri CRL 1098 to produce a compound with vitamin B12

activity (Taranto, Vera, Hugenholtz, Font, & Sesma, 2003). This

finding constitutes the first evidence of vitamin B12 produc-

tion in LAB. Subsequently, Santos et al. (2007) confirmed that

the corrinoid produced by this LAB strain under anaerobic

conditions is a Coa-[a-(7-adenyl)]-Cob-cyanocobamide or

pseudovitamin B12 Recently, Molina, Medici, Taranto, and

Font de Valdez (2008b) demonstrated that the corrinoid pro-

duced by L. reuteri CRL 1098 is biologically active and effective

in preventing the development of pathologies caused by

nutritional vitamin B12 deficiency in both pregnant mice and

their weaned young. Besides this attractive functional prop-

erty, this strain also has particular technological and probiotic

characteristics, e.g., easy and low-cost biomass production;

ability to grow on different food substrates; capacity to bear

environmental stress and reduce cholesterol levels (Taranto,

Font de Valdez, & Perez-Martinez, 1999, 2006).

The aim of this study was to develop a soy-bean-based

functional food using L. reuteri CRL 1098 – a cobalamin pro-

ducer lactobacilli – and to evaluate the efficiency of the bio-

fortified soymilk obtained to prevent the symptoms produced

by nutritional vitamin B12 deficiency in pregnant females and

their weaning offspring by using a previously standardized

experimental murine model (Molina, Medici, Taranto, & Font

de Valdez, 2008a).

2. Materials and methods

2.1. Microorganisms and growth conditions

The strain used in this study, L. reuteri CRL 1098, belongs to

Centro de Referencia para Lactobacilos culture collection

(CRL) (CERELA-CONICET, Tucuman, Argentina). Before experi-

mental use, cultures were grown in sterile MRS broth (De

Man, Rogosa, & Shape, 1960) and incubated at 37 �C for 16 h.

2.2. Preparation of fermented soymilk

A commercial soymilk (SM) (kindly provided by Refinerıa de

Maiz, UNILEVER) was used as a substrate. The composition

(per 100 ml) is as follows: protein, 2.6 g; carbohydrates, 4 g;

lipids, 1.5 g; fibre, 0.6 g; Ca, 48 mg; Fe, 0.84 mg; P, 48 mg, and

Mg, 18 mg; pH 7.3. Sterile SM (115 �C for 20 min) was cooled

to 37 �C, inoculated (1%, v/v) with L. reuteri CRL 1098, and incu-

bated at 37 �C for 6 h. The fermented SM had a final pH of 6.8

and a total colony counts of 1.6 · 107 cfu/ml. The concentra-

tion of vitamin B12 in the SM fermented was determined with

a quantitative bioassay that is used for the assessment of B12

in food (Kelleher & Broin, 1991). Briefly, L. delbrueckii subsp. lac-

tis ATCC 7830, a strain that requires cobalamin, was used to

evaluate this vitamin content in the fermented and unfer-

mented SM. Quantification analyses using a cyanocobalamin

standard curve indicated that the fermented SM contains

approximately 20 lg of cobalamin/litre. The vitamin was not

detected in the unfermented SM (control).

2.3. Animals

Six-week old pregnant female BALB/c mice (fourteen-days

pregnancy calculated from the first contact with the male) ob-

tained from the closed colony of the breeding unit kept at the

CERELA Institute (San Miguel de Tucuman, Argentina) were

individually housed in plastic cages (20 · 30 · 15 cm) and

maintained at 20 ± 2 �C with a 12-h light/dark cycle.

The animals were randomly allocated to four main groups

(each of five mice) as follows: B12-deficient females that

received a B12-deficient diet (DF group); B12-sufficient females

that received a B12-deficient diet plus soymilk supplemented

with 1.3 lg per kg of diet of commercial vitamin B12 (Parafarm,

Buenos Aires, Argentina) (CF group, control group); B12-deficient

females that received a B12-deficient diet plus non-fermented

soymilk (SF group); B12-deficient females that receiving a B12-

deficient diet plus soymilk fermented with L. reuteri CRL 1098

(107 cells/day/mouse) (SRF group). The B12-deficient diet used

in this study was provided by Biomedical Inc/ ICN (Irvine, CA,

USA). The animals were allowed free access to the diets and

water for 30 days from the middle of gestation (day 11th from

mating) up to weaning (day 21st after offspring birth).

The administration of soymilk (B12-supplemented, fer-

mented, and non-fermented) to females was carried out by

gavage (0.5 ml/day/mice).

The females belonging to the CF and SRF groups gave birth

to ca. ten young (CY and SRY group, respectively) while the fe-

males in the DF and SF groups gave birth to ca. five B12-deffi-

cient young (DY and SY group, respectively). The young

remained with their mothers until weaning and were selected

at random for the studies without considering sex. Females

(DF, CF, SF and SRF groups) continued to receive their corre-

sponding diets during the suckling period. Feed intake

(5.2 ± 0.6 g feed/day) was similar in all groups. Offspring re-

ceived only maternal milk.

The body weight of the females was recorded at the begin-

ning of the feeding up to the weaning period. The body weight

of the young was determined at the end of weaning (21-day-

old young). Results were expressed in grams (g). All determi-

nations in females (mothers) and offspring were performed

in five and ten mice/group, respectively, for statistical valida-

tion. Determinations in the offspring were carried out during

the weaning period (21-days old young).

2.4. Blood and organ collection

At the end of the trials, the females in each group and their

corresponding offspring were anesthetized with an intraperi-

Table 1 – Effect of the oral administration of soymilkfermented with Lactobacillus reuteri CRL 1098 on thebody weight of murine females (A) and their corre-sponding offspring (B).

Experimental group Body weight (g)

A DF 24.3 ± 1.6a

CF 37.5 ± 0.6b

SF 28.7 ± 1.5a

SRF 36.2 ± 1.2b

B DY 8.9 ± 0.8a

CY 13.1 ± 1.1b

SY 9.6 ± 1.2a

SRY 12.5 ± 0.9b

Values are means ± SD. Females mouse group: n = 5 (each one);

young groups: n = 30 (each one). Values with different superscript

letters (a and b) indicate significant differences among groups

(p < 0.05).

DF, B12-deficient females; CF, females receiving a B12-deficient diet

plus soymilk supplemented with commercial vitamin B12 (control

group); SF, females receiving a B12-deficient diet plus non-fer-

mented soymilk; SRF, females receiving a B12-deficient diet plus

soymilk fermented with L. reuteri CRL 1098; DY, weaned young

coming from B12-deficient females; CY, weaned young coming from

B12-sufficient females; SY, weaned young coming from B12-deficient

females fed with unfermented soymilk; SRY, weaned young coming

from B12-deficient females fed with the soymilk fermented with L.

reuteri CRL 1098.

J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 8 3 1 – 8 3 6 833

toneal injection of ketamin (5%) – xylacin (2%) (2.0 ml/kg

animal weight; 20:1 v/v) (Bayer S.A) and bled by cardiac punc-

ture. Blood was transferred into tubes with ethlenediamine-

tetraacetic acid (EDTA) solution (anticoagulant) to determine

haematological parameters and into plastic centrifuge tubes

for determination of vitamin B12 by immunoassay.

Freshly excised small intestine was removed and pro-

cessed for paraffin inclusion following the technique devel-

oped by Saint-Marie (1962).

2.5. Haematological parameters and serum vitamin B12

levels determination

Haematokrit (Hto) values and number of leukocytes and red

blood cells were determined by hematocytometric methods.

Differential cell counts were performed by counting 100 cells

in blood smears stained with May Grunwald-Giemsa. Haemo-

globin concentration was determined by colourimetric assays.

For examination of reticulocytes (% Ret), equal volumes

(100 ll) of blood and 1% brilliant cresol blue (BCB) were mixed

and incubated at 37 �C for 15 min. Blood sample smears were

prepared on glass slides with 5 ll of the cell suspension. The

Ret were counted under a microscope (1000· magnification) in

ten areas of the stained smears, corresponding to approxi-

mately 1000 red blood cells. Results are expressed as percent-

age of total red blood cells.

Vitamin B12 concentration was measured in serum sam-

ples by electrochemiluminiscence immunoassay (ECLIA) on

a Roche Elecsys 2010 automatic analyzer (Roche Diagnostics,

Basel, Switzerland) at the Laboratory for High Complex Clini-

cal Analysis – Quevedo S.R.L. (Tucuman, Argentina). Results

are expressed as pg/ml.

2.6. Histological studies

The small intestine was removed at the end of each treatment

and processed by modified Saint-Marie’s technique (1962).

Briefly, tissues were fixed in 10% formalin in phosphate saline

solution (PBS) for 48 h at room temperature and then dehydrated

in successive alcohols baths (40%, 50%, 70%, 96% and 100%) for

20 min each alcohol. Finally, samples were cleared by passing

through three consecutive xylene baths for 45 min each.

The tissue was embedded in paraffin at 56 �C for 3 h. Sec-

tioning was carried out as usual and tissue sections (3–4 lm)

were placed on glass slides.

2.7. Determination of IgA-producing cells in the smallintestine

The number of IgA-producing (IgA+) cells was determined on

histological slices of samples from the ileal region near Peyer’s

patches by direct immunofluorescence test (DIFT) (Vinderola,

Perdigon, Duarte, Farnworth, & Matar, 2006). The test was per-

formed by using a-chain specific FITC conjugated anti-mouse

IgA (Sigma–Aldrich, St. Louis, MO, USA). Deparaffinised histo-

logical samples were incubated with the antibody dilution

(1/100) in PBS (Phosphate Buffer Sodium 0.1 M, pH 7) solution

for 30 min at 37 �C. Samples were then washed three times

with PBS solution and examined by using a fluorescent light

microscope. Results were expressed as the number of IgA+ cells

(positive: fluorescent cell) per 10 fields (magnification 100·).

Results were the mean of three histological slices per animal.

2.8. Statistical analysis

A Student’s test was used to compare the data from vitamin

B12 deficient groups with the control group (females and off-

spring). Significant differences were considered at p < 0.05.

Experimental data were expressed as mean ± SD.

The Ethical Committee for Animal Care at CERELA ap-

proved all animal protocols. All assays complied with the cur-

rent laws of Argentina and followed the most recent

recommendations of the Federation of European Laboratory

Animal Science Associations.

3. Results

3.1. Animal weight

The body weight of mouse females and their corresponding

offspring are shown in Table 1. Females that received the L.

reuteri CRL 1098-fermented soymilk (SRF Group) and the B12

deficient diet showed a body weight similar to normal ani-

mals (CF group) that received soymilk supplemented with

commercial vitamin B12. In contrast, body weight was signif-

icantly lower in deficient females (DF group) and this smaller

development as a consequence of the B12-deficit was also

found in the females that received the unfermented soymilk

with the deficient diet (SF group).

On the other hand, the weaned young of females that re-

ceived the deficient diet and the fermented soymilk (SRY

group) reached a higher body weight than the offspring from

Table 3 – Vitamin B12 serum concentration in weanedyoung.

Experimental group Vitamin B12 (pg/ml)

DY 275 ± 35a

CY 735 ± 24b

SY 265 ± 13a

SRY 588 ± 24b

Values are means ± SD. Groups: n = 30 (each one). Values with dif-

ferent superscripts are significantly different (p < 0.05).

DY, weaned young coming from B12-deficient females; CY, weaned

young coming from B12-sufficient females; SY, weaned young

coming from B12-deficient females fed with unfermented soymilk;

SRY, weaned young coming from B12-deficient females fed with the

soymilk fermented with L. reuteri CRL 1098.

834 J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 8 3 1 – 8 3 6

females fed a deficient diet plus water or unfermented soy-

milk (DY and SY group, respectively). The values obtained

for the SRY group were statistically similar (p > 0.05) to those

found in normal young from control females (CY group) that

received soymilk supplemented with commercial vitamin B12.

3.2. Haematological determinations

The haematological values of females and their correspond-

ing offspring are shown in Table 2. Vitamin B12 deficiency

caused a decrease in the values of all haematological param-

eters evaluated (DF group) and this diminution was not

normalized when the animals received the unfermented soy-

milk with the deficient diet (SF group). On the contrary, the

females that received the deficient diet supplied with soymilk

fermented with L. reuteri CRL 1098 displayed a haematological

pattern similar to the one in the normal animals (CF group)

that received commercial vitamin B12. No statistically signifi-

cant differences (p > 0.05) in the total number of leukocytes

between the B12-deficient and the B12-sufficient groups

(females and young) were observed (data not shown).

3.3. Determination of serum vitamin B12 levels

The weaned young proceeding to the deficient females treated

with soymilk fermented with L. reuteri CRL 1098 (SRY group)

showed values of serum vitamin B12 similar (p > 0.05) to those

found in the offspring of the normal females that received soy-

milk supplemented with commercial vitamin B12 (Table 3). In

contrast, the consumption of unfermented soymilk by defi-

cient females did not normalize the serum vitamin B12 levels

in their corresponding offspring (SY group), which showed val-

ues similar to those found in the weaned young of females that

only received the deficient diet (Table 3).

3.4. Determination of IgA-producing cells in the smallintestine

A remarkable decrease in the number of IgA-producing (IgA+)

cells in the small intestine of deficient females in the DF and

Table 2 – Effect of the oral administration of soymilk fermenteparameters of murine females (A) and their corresponding off

Experimental group Hto (%

A DF 37.8 ± 2.6

CF 50.2 ± 5.6

SF 38.4 ± 3.2

SRF 48.6 ± 5.1

B DY 35.8 ± 3.3

CY 48.2 ± 2.5

SY 38.0 ± 1.9

SRY 45.2 ± 3.3

Values are means ± SD. Females mouse group: n = 5 (each one); young gro

b) indicate significant differences among groups (p < 0.05).

DF, B12-deficient females; CF, females receiving a B12-deficient diet plus so

females receiving a B12-deficient diet plus non-fermented soymilk; SRF, f

reuteri CRL 1098; DY, weaned young coming from B12-deficient females;

young coming from B12-deficient females fed with unfermented soymilk

soymilk fermented with L. reuteri CRL 1098.

SF groups was observed. In contrast, the females that received

soymilk fermented with L. reuteri CRL 1098 (SRF group)

showed IgA+ cells numbers similar to those of the normal fe-

males that received a commercial vitamin B12 (CF group). The

analysis of pups samples showed that though the SRY group

did not reach the values found in the control young (CY),

the same ones were significantly higher than those obtained

for the offspring belonging to the DY and SY groups (Table 4B).

Besides the decrease in the IgA+ cells number caused by

nutritional B12 deficiency, the small intestine of females in

the DF and SF groups showed severe alterations in the gut

mucosa (size of villi, oedema and leukocyte infiltrations). In

contrast, the females that received soymilk fermented with

L. reuteri CRL 1098 together with the deficient diet presented

a normal histological structure of the small intestine, similar

to the control animals that received soymilk supplemented

with commercial vitamin B12.

A similar behaviour was found in the corresponding young

groups.

4. Discussion

The development of functional foods (FF) constitutes an

important alternative to improve the quality of the diet

d with Lactobacillus reuteri CRL 1098 on the haematologicalspring (B).

) Ret (%) Hb (g dl�1)

a 2.7 ± 0.3a 11.8 ± 1.6a

b 4.8 ± 0.5b 15.0 ± 2.4b

a 2.8 ± 0.2a 12.1 ± 1.5a

b 4.2 ± 0.7b 14.5 ± 2.1b

a 2.2 ± 0.2a 8.2 ± 1.5a

b 4.3 ± 0.4b 12.0 ± 1.2b

a 2.3 ± 0.4a 8.5 ± 1.3a

b 3.9 ± 0.3b 10.8 ± 2.1b

ups: n = 30 (each one). Values with different superscript letters (a and

ymilk supplemented with commercial vitamin B12 (control group); SF,

emales receiving a B12-deficient diet plus soymilk fermented with L.

CY, weaned young coming from B12-sufficient females; SY, weaned

; SRY, weaned young coming from B12-deficient females fed with the

Table 4 – Effect of the oral administration of soymilkfermented with Lactobacillus reuteri CRL 1098 on IgAproducer cells of murine females (A) and their corre-sponding offspring (B).

Experimental group Number of IgA+ cells/10 field

A DF 66.4 ± 2.0a

CF 94.7 ± 3.4b

SF 63.2 ± 5,4a

SRF 87.0 ± 6.3b

B DY 38.0 ± 4.5a

CY 70.2 ± 5.0b

SY 35.7 ± 6,5a

SRY 49.8 ± 2.3b

Values are means ± SD. Females mouse group: n = 5 (each one);

young groups: n = 30 (each one). Values with different superscript

letters (a and b) indicate significant differences between groups

(p < 0.05).

DF, B12-deficient females; CF, females receiving a B12-deficient diet

plus soymilk supplemented with commercial vitamin B12 (control

group); SF, females receiving a B12-deficient diet plus non-fer-

mented soymilk; SRF, females receiving a B12-deficient diet plus

soymilk fermented with L. reuteri CRL 1098; DY, weaned young

coming from B12-deficient females; CY, weaned young coming from

B12-sufficient females; SY, weaned young coming from B12-deficient

females fed with unfermented soymilk; SRY, weaned young coming

from B12-deficient females fed with the soymilk fermented with L.

reuteri CRL 1098.

J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 8 3 1 – 8 3 6 835

considering that food selection can exert a positive influence

on well-being and health. The development of foods fer-

mented with selected Lactobacillus and Bifidobacterium strains

constitutes an important sector in the FF market. At present,

the development of dairy products using LAB probiotics is an

important topic with relevant implications in the industrial

and commercial areas. Nevertheless, one of the current

trends in the FF industry is the diversification of the sub-

strate used as a vehicle of the probiotic microorganisms to

replace the animal origin vehicles by others of vegetable ori-

gin. In this way, the use of oat (Angelov, Gotcheva, Kunche-

va, & Hristozova, 2006), millet (Lei, Friis, & Michaelsen, 2006)

and cereal extracts (Charalampopoulos, Pandiella, & Webb,

2003) as alternative fermentation substrates for BAL has

been reported. Within the potential vegetable vehicles, soy-

bean constitutes an interesting alternative considering its

valuable nutritional characteristics (Sarkar & Li, 2003; Squad-

rito et al., 2003). It is known that the organoleptyc and

health beneficial properties of soybean-based products can

be improved by using selected probiotic bacteria for the

development of fermented products (Wang, Yu, & Chou,

2006). However, soybean lacks essential micronutrients such

as vitamin B12. In the present work, we designed a soy-based

functional food with pseudovitamin B12-producing LAB (L.

reuteri CRL 1098) to restore the lack of vitamin B12 in this

vegetal food.

During pregnancy and lactation, micronutrient deficien-

cies are one of the major complications promoting infection

processes due to the high nutritional requirements needed

to support foetal and infant growth as well as maternal

metabolism. Since nutritional factors during early infancy

cause short and long term effects, the nourishment of moth-

ers, breast-fed babies and small children has great biological

importance. According to the experimental animal model de-

scribed by Molina et al. (2008a), a vitamin B12-deficient diet

during gestation and lactation causes several clinical and

haematological alterations in mouse dams and their corre-

sponding offspring. In this work, both the females and their

offspring that received unfermented soy milk presented the

typical symptoms of vitamin B12 depletion. Moreover, the

consumption of soymilk fermented with L. reuteri CRL 1098

for mice feeding with a B12 deficient diet prevented the devel-

opment of all manifestations that characterize this nutri-

tional deficiency. The females fed with the fermented

product and their offspring showed absence of anaemia and

normal body development similar to normal animals. These

results would demonstrate that the soymilk was bio-fortified

in situ and that its consumption had a biologically positive ef-

fect on the host.

On the other side it has also been established that nutri-

tional deficiency is commonly associated with an impaired

immune response (Jayarajan & Daly, 2011). Non-specific

mechanisms as intestinal microbiota balance, anatomical

barriers (mucosa and ephitelium) and secretory substances

(lysozymes and mucus) are affected by malnutrition (Erick-

son, Medina, & Hubbard, 2000). Some authors (Gauffin Cano

& Perdigon, 2003; Maldonado Galdeano et al., 2011) reported

that some of these alterations can be corrected by the admin-

istration of selected LAB strains. In this work we demon-

strated that the administration of soymilk fermented with L.

reuteri CRL 1098 reduced the histological and immunological

intestinal alterations caused by B12 nutritional deficiency

obtaining IgA+ cells values close to the control animals in fe-

males receiving the fermented soymilk and their respective

weaned young. These results would prove the bioavailability

of the vitamin produced by this strain.

Considering that actually the consumption of soybean is

important and that LAB are GRAS (generally regarded as safe)

microorganisms, the design of a soybean product bio-fortified

with vitamin B12 will contribute to modify the nutritional hab-

its of the population and to diversify the local diet, obtaining

all the advantages of a complete vegetal food.

Acknowledgement

This project was supported by grants of CONICET, FONCyT

and CIUNT from Argentina.

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