Bioaccessible antioxidants in milk fermented by Bifidobacterium longum subsp. longum strains

Abstract

Bifidobacterium longum subsp. longum is among the dominant species of the human gastrointestinal microbiota and could thus have potential as probiotics. New targets such as antioxidant properties have interest for beneficial effects on health. The objective of this study was to evaluate the bioaccessibility of antioxidants in milk fermented by selected B. longum subsp. longum strains during in vitro dynamic digestion. The antioxidant capacity of cell extracts from 38 strains, of which 32 belong to B. longum subsp. longum, was evaluated with the ORAC (oxygen radical absorbance capacity) method. On the basis of screening and gene sequence typing by multilocus locus sequence analysis (MLSA), five strains were chosen for fermenting reconstituted skim milk. Antioxidant capacity varied among the strains tested (P = 0.0009). Two strains of B. longum subsp. longum (CUETM 172 and 171) showed significantly higher ORAC values than the other bifidobacteria strains. However, there does not appear to be a relationship between gene sequence types and antioxidant capacity. The milk fermented by each of the five strains selected (CUETM 268, 172, 245, 247, or PRO 16-10) did not have higher initial ORAC values compared to the nonfermented milk samples. However, higher bioaccessibility of antioxidants in fermented milk (175-358%) was observed during digestion.

Figure 1

Antioxidant capacity of B. longum subsp. longum strains paired with the MLSA dendrogram. On the left, Jukes-Cantor neighbor-joining dendrogram constructed using the concatenated sequences of five loci (mutT1, ahpC, trx, nrdA, and ppk). Strains marked with a blue dot are the strains selected for milk fermentation. The length of the branches expressed in units of substitutions per site of the sequence alignment is indicated by the scale bar. On the right, oxygen radical absorbance capacity (ORAC) values correspond to the weighted means determined by ANCOVA. The error bar represents the standard error (SE).

Figure 2

Survival curve during in vitro digestion (TIM-1) of fermented milk with B. longum subsp. longum CUETM 172 (a), CUETM 247 (b), CUETM 245 (c), CUETM 268 (d), and PRO 16-10 (e). The cell concentrations were determined by viable counts in CFU/mL (solid line) and by PMA-qPCR in viable cells/mL (dashed line). Samples were taken in gastric (▲) and duodenal (■) compartments. Empty symbols indicate that only one value was obtained. The limit of detection of PMA-qPCR was 3.51 log of viable cells/mL. The error bars represent the standard deviation.

Figure 3

Survival rate of the five strains used for milk fermentation during in vitro digestion (TIM-1). The viable cells were determined by PMA-qPCR and by viable counts (VC) in the effluent after each hour of digestion. The remaining cells in the jejunum and ileum residue after 300 min of digestion were included in the survival rate. The results are presented relative to the total cells in the fermented milk at the start (% of intake). The error bars represent the standard deviation.

Figure 4

Bioaccessibility of antioxidants (in dialysates) evaluated with oxygen radical absorbance capacity (ORAC) during in vitro digestion (TIM-1) of fermented milk (300 g) by B. longum subsp. longum CUEMT 172 (▲), CUETM 268 (■), CUETM 245 (●), CUETM 247 (X), and PRO 16-10 (♦). (a) The cumulative quantity of bioaccessible antioxidants is expressed in μmol Trolox equivalent (TE). (b) The bioaccessibility of antioxidants expressed as a percentage of intake (antioxidants in 300 g of fermented milk before digestion). The error bars represent the standard deviation.