Almond milk fermented with different potentially probiotic bacteria improves iron uptake by ...

13 Pages·2015·0.79 MB·English

International Journal
of Food Studies
OFFICIAL JOURNAL OF THE ISEKI_FOOD ASSOCIATION

ISSN: 2182-1054
Almond milk fermented with different
potentially probiotic bacteria improves iron
uptake by intestinal epithelial (Caco-2) cells

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DOI : 10.7455/ijfs/4.1.2015.a4
International Journal of Food Studies IJFS April 2015 Volume 4 pages 49–60
Almond milk fermented with different potentially probiotic
bacteria improves iron uptake by intestinal epithelial (Caco-2)
cells
Neus Bernata*, Maite Cha´fera, Amparo Chiralta, Jose´ Moise´s Laparrab, and
Chelo Gonza´lez-Mart´ıneza
a
Instituto de Ingenier´ıa de Alimentos para el Desarrollo. Universitat Polit`ecnica de Val`encia, Camino de Vera
s/n, 46022 Valencia, Spain
bMicrobial Ecophysiology and Nutrition Laboratory. Instituto de Agroqu´ımica y Tecnolog´ıa de Alimentos
(CSIC). Av. Agust´ın Escardino, 7. 46980 Paterna, Valencia, Spain
*Corresponding author
[email protected]
Tel: [phone]
Fax: [phone]
Received: 6 Mars 2014; Published online: 18 April 2015
Abstract
Newfermentedalmondmilksweredeveloped,usingdifferentpotentiallyprobioticbacteria,inorder
to meet the current demand for healthy, versatile non-dairy products. An in vitro digestion/Caco-
2 cell model was used to evaluate the effect of both non-fermented and fermented almond milks on
the mitochondrial enzymatic activities of enterocytes. Moreover, macrophages were challenged with
the in-vitro digested samples and the production of pro-inflammatory biomarkers TNF-α and IL-6
was quantified. Enzymatic activities of cell cultures seemed to be stimulated by the exposure to both
fermentedandnon-fermentedalmondmilks. Bothbiomarkersdecreased(p<0.05)infermentedalmond
milkswitheitherB.bifidum orB.longum. Resultsshowedthatfermentedalmondproductsfavoredthe
energeticmetabolismofenterocytesandhadalowerinflammatoryresponsethannon-fermentedalmond
milk, suggesting its benefits for the management of allergies/intolerances. Moreover, the fermentation
process enhanced the uptake of iron by Caco-2 cells, especially when using L. rhamnosus and either
B. bifidum or B. longum as starters, thus improving the product bioactivity. Therefore, new non-
dairy fermented products with functional properties were developed, which might be positioned as
alternativestocow-milkproductsforsensitizedgroupsofpopulation(allergicand/orintoleranttocow
milk or anemic population, among others).
Keywords: Almond milk; Fermentation; Probiotics; Iron availability; Inflammation
1 Introduction to a reduced exposure to a variety of microbes,
which, early in life, is a crucial factor in the
The current alarming increase in the incidence development of allergies (Bj¨orkst´en, 2009). In-
of allergic diseases in both children and adults deed, several prospective follow-up studies have
in developed countries has been attributed to found that alterations in gut microbiota precede
the so-called “hygiene hypothesis”; this theory allergy development (Kalliom¨aki, 2010). Hence,
suggests that the increased level of hygiene in recentclinicalallergyresearcheshaveprincipally
both the environment and the food supply leads focused their attention on the manipulation of
Copyright '2015 ISEKI-Food Association (IFA) 10.7455/ijfs/4.1.2015.a4
50 Bernat et al.
gut microbiota composition (Kalliom¨aki et al., 2006), which might improve the bioavailability
2010). Nowadays, probiotics, prebiotics and of dietary iron. As it has been reviewed,
synbiotics (combination of pre- and probiotics) although non-heme iron is easily oxidized (not
are considered as good tools with which to bioavailable) due to the rise of pH in the
elicit changes in the gut biomass composition, lumen, the presence of reductants from food can
since they can improve and stimulate beneficial maintain this micronutrient in its reduced form
gut microflora, among other effects that are and, therefore, positively improve its absorption
beneficial for the health. Since the late 1990s, (Miret, Simpson, & McKie, 2003). Moreover,
over 30 randomized clinical trials have been almond milk is considered an appropriate alter-
published, in which probiotics have been used native to cow-milk, since, besides the healthy
either in the treatment or prevention of allergies lipid profile, it has a low ratio of Na/K and a
(mainly atopic diseases) (Kalliom¨aki et al., balanced ratio of Ca/P (Luengo, 2009).
2010). Hence, considering the high prevalence of Nevertheless, with regards to the inflammatory
atopic disease in childhood in the industrialized response, there is lack of data concerning nut
countries (Anandan, Nurmatov, van Schayck, & beverages, and least when it comes to derivative
Sheikh, 2010), the use of yoghurt-type foods as fermented products. Only recently has data
carriers of probiotics and/or synbiotics would appeared on almonds (Rajaram, Connell, &
be helpful as a means of attaining the either Sabate, 2010), in which the authors studied
preventive or prophylactic treatment in this whether, in addition to the lowering of blood
targeted population. Moreover, despite the lipids, monounsaturated fat-rich almonds influ-
little known and untrustworthy concept of enced other coronary heart disease risk factors,
functional food, consumers are familiar with such as inflammation. The study concluded
yoghurt-type products and consider them as that incorporating about 68 g almonds in a 2000
healthy (Annunziata & Vecchio, 2011), which kcal cholesterol-lowering diet decreased serum
would facilitate the inclusion of such functional E-selectin (molecules which are indicative of
fermented products in their diets. endothelial dysfunction) and C-reactive protein
The immunomodulatory effects of fermented (sensitive marker of inflammation that, in high
dairy-milks, casein hydrolysates and soy- concentrations, is strongly linked with coronary
beverage caused by lactic acid bacteria have events, stroke and peripheral vascular disease)
been widely reported (Baroja, Kirjavainen, in healthy men and women.
Hekmat, & Reid, 2007; Sutas et al., 1996; Although around 50% of almond composition is
Wagar, Champagne, Buckley, Raymond, & fat, intakes of 7g per day of this nut have been
Green-Johnson, 2009). However, in addition shown to reduce low-density lipoprotein choles-
to the allergenic proteins, both matrices might terol concentration by 1% (Sabat´e, Haddad,
provoke iron deficiencies in infants and toddlers. Tanzman, Jambazian, & Rajaram, 2003) and up
On the one hand, the calcium together with to84gperdaycanbeconsumedwithoutweight
the casein provided by cow-milk are seen to gain (Chen et al., 2006). In addition, these nuts
inhibit the absorption of dietary non-heme iron, havealowglycemicindex(theydonotadversely
in addition to the intestinal blood loss observed impact insulin sensitivity) (Chen et al., 2006)
in approximately 40% of infants during feeding and have been found to possess prebiotic effects,
with cow-milk and/or its derivatives (Agostoni since they stimulated the growth of gut bifi-
& Turck, 2011). On the other hand, soya-based dobacteria and Eubacterium rectale (Mandalari,
products contain phytates, which negatively Nueno-Palop, Bisignano, Wickham, & Narbad,
interfere in the absorption of iron, among other 2008). Hence, taking into account the health
minerals (Artazcoz, 2007). benefitsofalmondintake, almondmilkmightbe
By contrast, almond milk has not been reported considered as a good food matrix with which to
to interfere negatively in iron absorption. In- obtain healthy fermented products. Moreover,
deed, almonds have high anti-oxidant activity if the fermentation process is carried out by
owing to the α-tocopherol and polyphenolic potentially probiotic bacteria, the developed
constituents (Chen, Lapsley, & Blumberg, fermented product could be useful as a means
IJFS April 2015 Volume 4 pages 49–60
Fermented almond milks improve bioavailability of iron 51
of preventing some immunomodulatory diseases, perature).
such as allergies. Lactobacillus rhamnosus CECT 278, Lactobacil-
The Caco-2 cell line, commonly used in con- lus plantarum 3O9, Bifidobacterium bifidum
junction with in vitro digestion techniques, is CECT870,Bifidobacteriumlongum CECT4551,
a useful model for studying intestinal human Streptococcus thermophilus CECT 986, Lacto-
iron uptake, which it allows to occur simulta- bacillusdelbrueckii subs. Bulgaricus CECT4005
neously with food digestion, and is generally were used as starters, pure or mixed, as shown
regarded as the best available intestinal cell inTable2. Allthebacteriawerepurchasedfrom
model for studying the mechanisms associated CECT (Paterna, Spain), with the exception of
with vectorial iron transport (Glahn, Lee, the L. plantarum 3O9 strain, which was isolated
Yeung, Goldman, & Miller, 1998). In addition, from Guirra sheep milk and selected as a probi-
the macrophage-derived RAW 264.7 cell line otic in previous studies (Amorocho Cruz, 2011).
expresses key genes and proteins of principal For the preparation of starters inoculum, the
pathways for the production of regulatory strains were independently incubated for 24 h
cytokines (Novak, Babcock, Jho, Helton, & in their selective broths and then centrifuged at
Espat, 2003) and constitutes a cell model used 100 g (Medigriger-BL-S, JP-Selecta; Barcelona,
worldwide and a useful tool with which to study Spain) for 10 min at 4 ◦C to re-suspend the pel-
the inflammatory response(s) and metabolic let in PBS-1x buffer (10 mmol/L phosphate, 137
activity promoted by food-derived components mmol/L NaCl, 2.7 mmol/L KCl, pH 7.4) until
while still maintaining a rapid and inexpensive reaching strain concentrations of 108 cfu/mL.
system (Deepika, Rastall, & Charalampopoulos, For each formulation, 1% (v/v) of starter sus-
2011; Kabeerdoss et al., 2011). pension was added to the almond milk and sub-
The aim of this study, therefore, was to evaluate sequently incubated at 37 ◦C until pH values of
whether almond milk, fermented with different 4.4-4.6werereached,whichwascontrolledbyus-
potential probiotic bacteria, affects both the ing a GLP 21+ pH-meter (Crison Instruments
energetic metabolism in intestinal cells and the S.A.; Barcelona, Spain). The fermented samples
production of pro-inflammatory biomarkers in were frozen and stored at -22 ◦C prior to anal-
order to gain insights into the potential benefits ysis. A non-fermented almond sample was used
of the designed products for the consumer’s gut as a control (C).
health.
2.2 Simulated gastrointestinal
2 Materials and Methods
digestion
2.1 Preparation and fermentation
The human gastrointestinal digestion process
of almond milk was simulated by using porcine pepsin (800-
2, 500 units/mg protein), pancreatin (activity,
Almond milk was produced by soaking and 4 USP specifications) and bile, as previously de-
grindingalmonds(PrunusamygdalusL. cv. dul- scribedbyLaparra,Barbera,Alegria,Glahn,and
cis) supplied by Frutos Secos 3G S.L. (Valen- Miller (2009). All reagents were purchased from
cia, Spain). The extraction was carried out in Sigma-Aldrich Co. (St Louis, MO, USA). Prior
the Sojamaticfi 1.5 (Barcelona, Spain), equip- to the in vitro digestion, 1.5 mL aliquot of each
ment specifically designed for the production assayed sample was diluted in 5mL of a saline
of vegetable milks, using a nut:water ratio of solution (140 mmol/L NaCl, 5 mmol/L KCl ad-
8:100. The milky liquid obtained was then mi- justed to pH 3). For gastric digestion (pepsin in
crofluidized in a high pressure homogenizer (M- 0.1 mol/L HCl adjusted to pH 3; 1 h), samples
110P model; Microfluidics International Corpo- were placed on a rocking platform shaker in an
ration, Westwood, MA, USA) by applying 172 incubator (37 ◦C; 5% CO ; 95% relative humid-
2
MPa, sterilized (121 ◦C/15 min) and subse- ity). Theintestinaldigestion(pancreatin-bileex-
quentlycooleddownto37◦C(fermentationtem- tractin0.1mol/LNaHCO adjustedtopH6.9-7;
3
IJFS April 2015 Volume 4 pages 49–60
52 Bernat et al.
2 h) was carried out in the upper chamber of a tures grown in MEM averaged 4.2 ng/mg cell
two-chamber system in 6-well plates. protein. Thesampleswereanalyzedintriplicate.
The upper chamber was formed by fitting the
bottom of an appropriately sized Transwell in-
2.4 Mitochondria enzyme
sert ring (Corning B.V. Life Sciences, Amster-
activities
dam, The Netherlands) with a 15,000 molecu-
lar mass cut-off dialysis membrane (Spectra/Por
These activities were evaluated in Caco-2
2.1, Spectrum Medical, Gardena, CA, USA).
cell cultures by monitoring MTT (3-(4,5-
Aliquots (1.5 mL) of the gastrointestinal digest
dimethylthiazol-2-yl)-2,3-diphenyl tetrazolium
were loaded into the upper chambers and incu-
bromide) conversion on exposed cultures after
bated for 2 h. Afterwards, the inserts were re-
an incubation period of 3 h, following the
movedandthe dialysateswerediluted(1:4, v/v)
method described by Laparra et al. (2009). This
withculturemediaandincubatedwithintestinal
colorimetric method is based on the reduction
epithelial (Caco-2) or macrophage (RAW 264.7)
of the tetrazolium ring of MTT by mitochon-
cells.
dria dehydrogenases yielding a blue formazan
product, which can be measured spectropho-
2.3 Ferritin analysis in intestinal tometrically. For the assays, Caco-2 cells were
epithelial cell monolayer seeded at 50,000 cell/cm2 in 24-well culture
plates(Costar,Cambridge,MA,USA),andwere
grown with DMEM for 12 days. Control cells
Caco-2 cells were obtained from the American
(basal) exposed to digests containing enzymes
Type Culture Collection (Rockville, MD, USA)
but not samples were used throughout each
at passage 17 and used in experiments at pas-
assay. Four replicates were analyzed.
sages 33 to 38. Cells were maintained with
Dulbecco’s modified Eagle’s medium (DMEM)
(Gibcofi, Madrid, Spain) under conditions pre- 2.5 Analysis of pro-inflammatory
viously described by Glahn et al. (1998).
markers
Fortheassays,Caco-2cellswereseededat50,000
cell/cm2 incollagen-treated6-wellcultureplates
The inflammatory analyses were carried out
(Costar,Cambridge,MA,USA),andweregrown
following the method described by Novak et
with DMEM for 12 days. On the day prior to
al. (2003). For the assays, RAW 264.7 cells
the experiments, the DMEM medium was re-
were seeded at 50,000 cell/cm2 and were grown
placed by 2 mL of minimum essential medium
with Roswell Park Memorial Institute (RPMI)
(MEM) (Gibcofi, Madrid, Spain) and then the
medium (Gibcofi, Madrid, Spain) for 24 hours.
cells were returned to the incubator. 50 µmol/L
Tumor Necrosis Factor-α (TNF-α) and inter-
ofFeCl wereaddedtothedigestedalmondmilk
3 leukin 6 (IL-6) (eBioscience Ltd., Hatfield, UK)
samples and the ferritin formation by Caco-2
were determined by ELISA, following the man-
cells over a 24 h period was proportional to the
ufacturer’s instructions, on exposed RAW 264.7
cell iron uptake. A latex-enhanced turbidimet-
cell cultures after an incubation period of 3 h.
ric immunoassay (Ferritin-turbilatex; Spinreact,
Results were expressed as picograms per mL of
Girona, Spain) was used to measure the Caco-2
media. Four replicates were analyzed.
cell ferritin content, as Glahn et al. (1998) de-
scribed. The concentrations of ferritin were nor-
malizedbythedeterminationofthetotalprotein 2.6 Statistical analysis
content in cell cultures by using a microLowry
kit (TP0200) (Sigma-Aldrich, St. Louis, MO, Each of the experiments was conducted on four
USA). The control cells (basal), exposed to in independent replicates. A one-way analysis of
vitro digestions of control solutions containing variance (ANOVA) and the Tukey post hoc test
digestive enzymes but not sample, were moni- were applied. Statistical significance was estab-
tored throughout. Base-line cell ferritin in cul- lished at a confidence level of 95% for all the
IJFS April 2015 Volume 4 pages 49–60
Fermented almond milks improve bioavailability of iron 53
comparisons. SPSS v.15 software (SPSS Inc., 3.2 Bacterial fermentation effects
Chicago, IL, USA) was used for the statistical on TNFα and IL-6 production
analysis.
Figure 1 shows the Tumor necrosis factor
(TNF)-α and interleukin (IL)-6 production in
3 Results and Discussion macrophage (RAW 264.7 cells) cultures exposed
to digests of fermented milks. A non-fermented
almond milk was used as a control (C).
3.1 Almond milk and their Focusingonthecellstreatedwithdialyzablefrac-
fermented-derivative products tionsamplesnotfermentedbyusingbifidobacte-
ria (F1 to F5) (Table 2), fermentation with ei-
ther L. rhamnosus (F2) or L. plantarum (F3)
The use of high pressure homogenization (HPH)
decreased the TNF-α production (p< 0.05) by a
contributed to a better stability of the almond
similar amount with respect to the control (C).
milk, since this emergent microfluidization tech-
When the standard yoghurt bacteria was used
nology is able to reduce the size of fat glob-
(F1), the TNF-α production was similar to the
ule particles, greatly delaying the flocculation
control. Oppositeeffectswereobservedwhenthe
andcoagulationphenomena(LujanCapraetal.,
fermentation was developed by combining those
2009; Cruz et al., 2009).
lactobacilli with standard yoghurt bacteria (F4
The chemical composition of the almond milk
and F5, respectively), since the TNF-α produc-
used for fermentations, which is subsequently
tion increased (p< 0.05) compared to the con-
codified as control (C), is summarized in Table
trol (Figure 1). As regards the IL-6, and con-
1. Taking into account the nut:water ratio used,
trarytothatobservedinTNF-α,allsampleshad
these values are similar to those found in the lit-
a positive effect in the production of this pro-
erature (Yada, Lapsley, & Huang, 2011).
inflammatorymarkerwithrespecttothecontrol,
The initial pH of the almond milk (C) was 6.61
especially when the almond milk fermentation
±0.08. AsignificantdeclineinpHto4.60±0.02
was done by using mixed-cultures (F4 and F5).
after 20 h at 37 ◦C of incubation, took place in
These samples exhibited the lowest IL-6 concen-
thedevelopedproductsalongwithacorrespond-
trations (p< 0.05), showing 55-70% of inhibition
ing increase in acidity caused by fermentation.
(p< 0.05) of the initial IL-6 production induced
by non-fermented almond milk (C) (Figure 1).
Withrespecttothesamplesfermentedusingthe
Table 1: Chemical composition of almond milks
bifidobacteria (F6 to F11), all the cells exposed
used in the study. Values (mean ± standard de-
to those dialyzed samples exhibited a very low
viation) are expressed as grams per 100 mL of
TNF-α production in comparison to the con-
beverage.
trol (p< 0.05). When considering IL-6 produc-
Compound Concentration tion, different patterns were observed depending
on the type of bifidobacteria present in starter.
Dry matter 6.64 ± 0.5
As Figure 1 shows, the fractions from fermented
Lipids 3.96 ± 0.2
samples with B. bifidum (F6, F8 and F10) in-
Proteins 1.37 ± 0.03
duced cells to produce IL-6 amounts similar to
Total sugars 0.41 ± 0.002
that obtained in fermented samples with stan-
Ashes 0.325 ± 0.012
dard yoghurt bacteria (F1). However, with B.
Fiber 0.58*
longum (F7, F9 and F11) lower (p< 0.05) IL-6
concentrations were quantified, especially when
*Fiberconcentrationwasobtainedbysubtractingthe
it was combined with either standard yoghurt
dry matter content from the sum of the rest of com-
bacteria (F7) or L. plantarum (F11).
pounds shown.
TNF-α is a pro-inflammatory factor produced
by macrophages that exerts important effects on
IJFS April 2015 Volume 4 pages 49–60
54 Bernat et al.
Figure 1: Tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in macrophage (RAW 264.7
cells) cultures exposed to digests of fermented milks. C: non-fermented almond milk. Basal: cells not
exposed to samples.*a-e DifferentsuperscriptlettersindicatesignificantdifferencesbetweensamplesinTNF-α
production (p< 0.05). * w-z Different superscript letters indicate significant differences between samples in IL-6
production (p< 0.05).
Table 2: Microbial strains used to produce the different fermented almond milks.
Formulation Starters inoculum
C - - -
F1 - - S. thermophilus + L. delbrueckii
F2 L. rhamnosus - -
F3 L. plantarum - -
F4 L. rhamnosus - S. thermophilus + L. delbrueckii
F5 L. plantarum - S. thermophilus + L. delbrueckii
F6 - B. bifidum S. thermophilus + L. delbrueckii
F7 - B. longum S. thermophilus + L. delbrueckii
F8 L. rhamnosus B. bifidum -
F9 L. rhamnosus B. longum -
F10 L. plantarum B. bifidum -
F11 L. plantarum B. longum -
L.: Lactobacillus B.: Bifidobacterium S.: Streptococcus
IJFS April 2015 Volume 4 pages 49–60
Fermented almond milks improve bioavailability of iron 55
systemic inflammation and induces the produc- deed,previousinvitroandinvivostudiescarried
tion of other inflammatory cytokines such as IL- out by using these bacteria species also showed
6 orIL-8 (Hu, Kobayashi, Zenda, & Shimamura, positive effects in immunomodulatory responses
1997). The observed bacterial fermentation ef- (Laparra, Olivares, Gallina, & Sanz, 2012; La-
fects could have important consequences on the parra & Sanz, 2010). Nevertheless, despite the
intestinal barrier function because TNF-α plays results, the differences from different probiotic
a crucial role by increasing paracellular perme- strains used are yet to be investigated in detail.
abilityandimpairingtightjunctionfunctionality
(Maetal.,2004)andleukocyteinfiltrationinthe
3.3 Bacterial fermentation effects
intestinal wall (Hoffman, 2000). In addition, the
reduction in TNF-α production might also have on energetic metabolism of
importantphysiologicalconsequencespreventing intestinal cells
allergic inflammatory processes.
Almonds are known to have several nutritional Resultsofthepossibletoxicityoffermentedsam-
benefits, including that of lowering cholesterol ples in intestinal epithelial (Caco-2) cells, which
and protection against diabetes (Rajaram et al., was quantified by monitoring the mitochondrial
2010; Sabat´e et al., 2003). Furthermore, sci- enzyme (MTT test) activities, are shown in Fig-
entific studies have pointed to their potential ure 2. The intestinal epithelium constitutes
prebiotic and anti-inflammatory activities (Ra- the first physiological barrier to exogenous com-
jaram et al., 2010; Mandalari et al., 2008). Al- pounds and nutrient absorption. Mitochon-
mond lipids and carbohydrates available for fer- dria and endo-lysosomal enzyme activities were
mentation have been associated with increasing proven to be sensitive biomarkers of changes in
both the number of bifidobacteria strains and cellular metabolism in response to internalized
the short chain fatty acids (SCFA) content, es- food-derived compounds (Laparra et al., 2009;
pecially in butyrate concentrations (Mandalari Wu et al., 2010). This MTT assay showed
et al., 2008). Butyric acid has the potential that none of the dialysates exposed to cell cul-
to benefit colonic health, since it reduces hy- tures seem to cause toxic effects, as concluded
drogen peroxide in cells and maintains their in- from the fact that the MTT values were similar
tegrity (Scott, Martin, Duncan, & Flint, 2014). (p< 0.05) to those calculated for basal culture
Although bifidobacteria strains are not able to (cells not exposed to almond milk samples). As
produce butyrate, they synthesize other SCFA mentionedabove,possiblesynergismbetweenal-
such as acetate which can be used by other bac- mondconstituents(prebiotics)andstarterbacte-
teria (i.e. lactobacilli) in their metabolic route riamighthaveproducedcellprotectivebioactive
to synthesize this cells’ protective acid (Falony, compounds (i.e. butyrate) (Falony et al., 2006;
Vlachou, Verbrugghe, &DeVuyst, 2006). More- Scott et al., 2014) which prevented alterations
over, this butyric production may be enhanced oftheCaco-2mythocondriaandendo-lyososomal
in the presence of prebiotics (i.e. almond fibers) enzymatic activity.
(Falony et al., 2006; Scott et al., 2014). These Particularly, the MTT values showed that
facts are coherent and might explain the marked dialysates from samples fermented with L. plan-
positive effects observed in the inflammatory re- tarum 3O9 (F3) (Table 2) stimulated the en-
sponses induced by dialysates from samples in- ergetic cell metabolism, similar to that of non-
oculated with bifidobacteria. Nevertheless, the fermented almond milk (C) (p< 0.05). How-
active components above mentioned (SCFA and ever, when these bacteria were used in combina-
prebiotics) have not been analysed since they tionwithstandardyoghurtbacteria,theresulted
were beyond the objective of this study. dialysates (F4 and F5) did not have this signifi-
As the results suggest, the involvement of ei- cant stimulatory effect.
ther B. bifidum or B. longum in the develop- Both MTT results (Figure 2) and the ones ob-
mentoffermentedalmond-basedproductsmight tainedintheproductionofpro-inflammatorycy-
be of interest in order to reduce inflammatory tokinesbyimmunecells(Figure1)indicatedthat
intestinalprocessesofatargetedpopulation. In- almond fermented products might exert benefi-
IJFS April 2015 Volume 4 pages 49–60
56 Bernat et al.
Figure 2: MTT conversion percentages in Caco-2 cell cultures exposed to digests of fermented milks.
(C: non-fermented almond milk; Basal: cells not exposed to samples). * a,b Different superscript letters
indicate significant differences(p< 0.05) between samples.
cialeffectsonhumanguthealth,especiallywhen trices, such as carrot juice (Bergqvist, Andlid,
using standard yoghurt bacteria with B. longum & Sandberg, 2006), maize (Proulx & Reddy,
(F7) and L. rhamnosus with B. longum (F9) as 2007) or beans (Laparra, Tako, Glahn, & Miller,
starters. 2008). This study, hence, extends the bacterial-
mediatedpositiveeffectsonironuptakefromfer-
mented vegetable products, particularly those in
3.4 Iron uptake in the presence of
which the starter culture contains bifidobacte-
fermented almond milk ria strains. In addition, it has been reported
that almond extracts exhibited excellent metal
Ferritin concentrations in cell cultures exposed ion chelation efficacies (ability to maintain oli-
todifferentdialyzedfermentedalmondmilks are goelements such as iron in the reduced form
shown in Figure 3. Apparently, the fermenta- needed to be absorbed by the epithelial cells),
tion process improved the bioavailability of iron, owingtoitssourceofbioactivepolyphenolswith
since the resultant Caco-2 iron uptake in every antioxidant activity (Garrido, Monagas, Gomez-
fermented formulation was higher than that ob- Cordoves, & Bartolome, 2008; Wijeratne, Abou-
tained in the cells exposed to non-fermented al- Zaid, & Shahidi, 2006). These almond-derived
mond milk dialysates (C) (p< 0.05). In par- components with functional characteristics may
ticular, samples fermented with L. rhamnosus, also be present in the fermented samples and
with either B. bifidum (F8) or B. longum (F9), might explain, at least in part, the effects ob-
were the ones which induced the most iron up- served.
take(p<0.05). Previousstudieshavealsoshown Therefore, as Figure 3 suggested, fermented
the in vitro enhancing effect of probiotic bacte- almond milk may provide health benefits to
ria on iron uptake in fermented vegetable ma-
IJFS April 2015 Volume 4 pages 49–60
Fermented almond milks improve bioavailability of iron 57
Figure 3: Ferritin concentration in Caco-2 cultures exposed to digested fermented milks with added
FeCl (50 µmol/L). (MEM: Minimum Essential Medium; C: non-fermented almond milk; Basal: cells
3
not exposed to samples).* a,e Different superscript letters indicate significant differences (p< 0.05) between
samples.
consumers owing to its ability to increase the lialcells,especiallywhenthisvegetablemilkwas
bioavailability of dietary iron. Moreover, this fermented with either standard yoghurt bacte-
positive effect observed could have important ria and B. longum CECT 4551 or L. rhamnosus
consequences as fermented almond milk might CECT278andB.longum. Moreover,somecom-
preserve the nutritional iron status in the pedi- binationsofspecificstrainshadmarkedlysignifi-
atric community which appears to be the most cantpositiveeffectsontheironuptakebyintesti-
susceptible population to the negative effects of nalepithelialcellsthatcouldhelptoimprovethe
cow-milk. Furthermore, the intake of this type nutritionalstatusoftargetedconsumers. Inpar-
ofproductcouldreduceallergiesandintolerances ticular, samples inoculated with L. rhamnosus
derivedfromtheconsumptionofcow-milkbythis CECT 278 and either B. bifidum CECT 870 or
population (Agostoni & Turck, 2011). B. longum CECT4551exhibitedthehighestfer-
ritin concentrations in Caco-2 cultures. The ob-
tainedresultsalsosuggestanimprovementinthe
4 Conclusions bioactivity of almond milk due to fermentation;
nevertheless, theidentificationofbiologicallyac-
This study has shown that almond milk fer- tive components is needed and will provide fur-
mented with potentially probiotic bacteria ex- ther insights into the potential nutritional and
erted positive immunomodulatory effects on healthbenefitsoffermentedalmond-basedprod-
macrophages and did not impair negative effects ucts. Tosumup,theresultssuggestthatalmond
on the energetic metabolism of intestinal epithe- milk fermented with potentially probiotic bacte-
IJFS April 2015 Volume 4 pages 49–60