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Inflammatory properties of almond milk fermented with potentially

probiotic bacteria. Bernat N1, Chafer M1, Chiralt A1, Sanz Y2, Gonzalez-Martínez C1 and Laparra JM2,

(1) Instituto de Ingeniería de Alimentos para el Desarrollo. Universidad Politecnica de Valencia, Camino

de Vera s/n, 46022 Valencia (Spain)

(2) Agrochemistry and Food Technology Institute (IATA-CSIC). Microbial Ecophysiology and Nutrition

Laboratory. Avd. Agustín Escardino, 7. 46980 Paterna, Valencia, (Spain)

ABSTRACT

A fermented product was developed by using almond “milk” produced by soaking and

grinding almonds cv. Marcona by using different potentially probiotic bacteria. An in

vitro digestion/Caco-2 cell model was used to evaluate the effect of not fermented

almond milk (NFAM) and the fermented products on the mitochondrial enzymes

activities (test MTT) of enterocytes. Otherwise, macrophages (RAW 264.7 cells) were

challenged with the digests and the production of pro-inflammatory biomarkers (TNFα

and IL-6) was determined by ELISA. A commercial milk-based infant formula (MBIF)

providing B. bifidus was tested for comparison.

MTT conversion values were increased in cell cultures exposed to almond milks,

fermented or not, but decreased in those incubated with the digests from MBIF. There

was a significant (P<0.05) decrease in the production of TNFα by macrophages

exposed to digests from fermented almond milks inoculated with B. bifidum CECT 870

(5.8 ng/ml) or B. longum CECT 4551 (4.2 ng/ml) relative to NFAM and MBIF, which

caused similar TNFα concentrations in cell culture supernatants (7.3-7.9 ng/ml).

Macrophage cultures exposed to NFAM exhibited the highest IL-6 production, and

MBIF produced IL-6 concentrations similar to almond milk inoculated with L.

plantarum. Almond milk inoculated with both bifidobacteria caused the lowest

production of IL-6.

The results indicate that fermented almond milk favours energetic cell metabolism

of enterocytes and had lower inflammatory potential than MBIF suggesting healthy

benefits from those in managing cow-milk allergy/intolerance.

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INTRODUCTION

Immunomodulatory effects of fermented soy- or dairy-milks and casein

hydrolysates by lactic acid bacteria have been widely reported [1, 2]. However, there is

a lack of data concerning milks from vegetal origin, and only recent data appeared

concerning almond seeds [3, 4].

The aim of this study was to evaluate whether fermented almond milks by different

potential probiotic bacteria affect energetic metabolism in intestinal cells and the

production of proinflammatory biomarkers by immunocompetent cells to gain insights

about the potential benefits for the consumer gut health.

MATERIAL AND METHODS

Preparation and fermentation of almond beverage. Almond beverage was

produced by soaking and grinding Marcona almonds. The aqueous extraction was

carried out in Sojamatic 1.5 equipment (Sojamatic®; Barcelona, Spain). The milky

liquid obtained was then ultra-homogenized at 172 MPa (M-110P model; Microfluidics

International Corporation, USA), heated at 121 ºC/15 min and fermented at 37 ºC/24 h

with L. rhamnosus CECT 27 (s2), L. plantarum CECT 220 (s3), L. delbrueckii subs.

Bulgaricus and S. thermophilus inoculated with cell suspensions (107-8 cells/ml) of

either B. bifidum CECT 870 (s6) or B. longum CECT 4551 (s7).

Simulated gastrointestinal digestion. The human gastrointestinal digestion process

was simulated as previously described [5]. As controls, non-fermented almond milk

(NFAM) and a commercial milk-based infant formula (MBIF) were used.

Mitochondrial and Lysosomal enzyme activities. For the experiments intestinal

epithelial (Caco-2) cells were used (5 days post-seeding) after an incubation period of 3

h. Mitochondrial activities were investigated by monitoring MTT (3-(4,5-

dimethylthiazol-2-yl)-2,3-diphenyl tetrazolium bromide) conversion. Lysosomal

activities were investigated by using the neutral red (NR) (toluylene red; 3-amino-7-

dimethylamino-2-methylphenazine hydrochloride) uptake assay. Control cells exposed

to digests containing enzymes but not samples were used throughout each assay.

Analysis of pro-inflammatory markers. TNFα, IL-1β, and IL-6 (eBioscience)

were determined by ELISAs, according to the instruction of the manufacturers, on

exposed RAW 264.7 cell cultures after an incubation period of 3 h.

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Statistical analysis. Each of the experiments was conducted in four independent

replicates. One-way analysis of variance (ANOVA) and the Tukey post hoc test were

applied. Statistical significance was established at P<0.05 for all comparisons. SPSS

v.15 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis.

RESULTS AND DISCUSSION

A significant decline in pH from 6.6 to 4.6 after 20 h at 37ºC of incubation with a

corresponding increase in titratable acidity due to fermentation occurred in the

developed product.

Bacterial fermentation effects on TNFα production. Cells treated with the

dialyzable fractions from samples fermented with either L. rhamnosus CECT 278 (s2)

or L. plantarum CECT 220 (s3) did not change (P>0.05) TNFα production from

controls (Fig. 1). Otherwise, only cells exposed to dialysates from samples fermented

with L. rhamnosus CECT 278 exhibited lower IL-6 concentrations than controls.

Fermentation with the bifidobacteria (s6, s7) had a positive effect decreasing (P<0.05),

up to 50%, the production of TNFα relative to controls. When considering IL-6

production, cell cultures challenged to fermented samples inoculated with either B.

bifidum CECT 870 (s6) or B. longum CECT 4551 (s7) exhibited lower production of IL-

6 than those inoculated with lactobacilli.

The bacterial fermentation effects observed could have important consequences on

the intestinal barrier function because TNFα plays crucial roles increasing paracellular

permeability impairing tight junction functionality [6] and leukocytes infiltration in

intestinal wall [7]. In addition, IL-6 is a key mediator of a multifunctional

proinflammatory cytokines, and the major cytokine produced by activated mast cells.

Almonds are known to have several nutritional benefits and it has been recently

suggested their potential prebiotic, increasing numbers of bifidobacteria, associated to

almond lipids available for fermentation [8]. This effect could explain, at least in part

the observed results.

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Cont s2 s3 s6 s7 MBIF

TNFα

(pg/

mL)

0

2000

4000

6000

8000

10000

IL-6

(pg/

mL)

0

50

100

150

200

250

300

TNFa IL-6

Figure 1. Production of pro-inflamatory markers, TNFα and IL-6, on exposed RAW

264.7 cell cultures to digests from non-fermented (control), commercial milk-based

infant formula (MBIF) and fermented samples with L. rhamnosus (s2), L. plantarum

(s3), yogurt cultures + B. bifidum (s6), yogurt cultures + B. longum (s7).

Bacterial fermentation effects on energetic metabolism of intestinal cells. The

cytotoxicity of ferments to intestinal epithelial (Caco-2) cells was determined,

monitoring the mitochondrial enzyme (test MTT) and endo/lysosomal (test NR)

activities (Fig. 2). Both metabolic assays showed that none of the dialysates exposed to

cell cultures caused toxic effects as concluded from MTT and NR values (%) similar

(P>0.05) or higher (P<0.05) than those calculated for controls.

Only were detected significant (P<0.05) differences in mitochondrial enzyme

activities coupled to the cellular energetic metabolism. MTT values showed that

dialysates from samples fermented with L. rhamnosus CECT 278 (s2) or L. plantarum

CECT 220 (s3) had a positive effect on energetic cell metabolism similar to that of non

fermented almond milk. These results evidence the non cytotoxic effect to intestinal

cells of the fermented almond milks. Cell cultures exposed to dialysates from samples

fermented with either B. bifidum CECT 870 (s6) or B. longum CECT (s7) exhibited

similar MTT conversion percentages suggesting similar effects on energetic cell

metabolism. Interestingly, the stimulatory capacity of fermented almond milks on

energetic cell metabolism resulted higher than observed in cell cultures challenged to

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the dyalysates from the MBIF. Increases in MTT conversion values have been

associated to the production of reducing equivalents preserving alterations in the

cellular redox status [9]. It has been reported that almond extracts are a source of

bioactive polyphenols with antioxidant activity [10]. These almond-derived components

with functional characteristics could also be present in the fermented samples and may

explain the effects observed.

Basal NFAM s2 s3 s6 s7 MBIF

% o

f bas

al c

ultu

res

80

90

100

110

120

130

140

150

MTT NR

Figure 2. MTT conversion and neutral red (NR) uptake values in intestinal epithelial

(Caco-2 cells) exposed to digests from non-fermented almond milk (NFAM),

commercial milk-based infant formula (MBIF) and fermented samples with

L.rhamnosus (s2), L. plantarum (s3), yogurt cultures + B. bifidum (s6), yogurt

cultures + B. longum (s7).

CONCLUSIONS

The results indicate that fermented almond milk favours energetic cell metabolism

of enterocytes and had lower inflammatory potential, when inoculated with with either

B. bifidum CECT 870 or B. longum CECT 4551, than the commercial MBIF containing

bifidobacteria. These results suggest health benefits from fermented almond milks that

may be helpful in managing cow-milk allergy/intolerance.

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