Patricia Savard

Assessment of Probiotic Viability during Cheddar Cheese Manufacture and Ripening Using Propidium Monoazide-PCR Quantification

The use of a suitable food carrier such as cheese could significantly enhance probiotic viability during storage. The main goal of this study was to assess viability of commercial probiotic strains during Cheddar cheesemaking and ripening (4–6 months) by comparing the efficiency of microbiological and molecular approaches. Molecular methods such as quantitative PCR (qPCR) allow bacterial quantification, and DNA-blocking molecules such as propidium monoazide (PMA) select only the living cells’ DNA. Cheese samples were manufactured with a lactococci starter and with one of three probiotic strains (Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus rhamnosus RO011, or Lactobacillus helveticus RO052) or a mixed culture containing B. animalis subsp. lactis BB-12 and L. helveticus RO052 (MC1), both lactobacilli strains (MC2), or all three strains (MC3). DNA extractions were then carried out on PMA-treated and non-treated cell pellets in order to assess PMA treatment efficiency, followed by quantification using the 16S rRNA gene, the elongation factor Tu gene (tuf) or the transaldolase gene (tal). Results with intact/dead ratios of bacteria showed that PMA-treated cheese samples had a significantly lower bacterial count than non-treated DNA samples (P < 0.005), confirming that PMA did eliminate dead bacteria from PCR quantification. For both quantification methods, the addition of probiotic strains seemed to accelerate the loss of lactococci viability in comparison to control cheese samples, especially when L. helveticus RO052 was added. Viability of all three probiotic strains was also significantly reduced in mixed culture cheese samples (P < 0.0001), B. animalis subsp. lactis BB-12 being the most sensitive to the presence of other strains. However, all probiotic strains did retain their viability (log 9 cfu/g of cheese) throughout ripening. This study was successful in monitoring living probiotic species in Cheddar cheese samples through PMA-qPCR.

Bioaccessibility and digestive stability of carotenoids in cooked eggs studied using a dynamic in vitro gastrointestinal model.

Among dietary carotenoids, lutein and zeaxanthin are known to protect against age-related macular degeneration, a leading cause of irreversible vision loss in the elderly. Egg yolk is rich in lutein and zeaxanthin, however, the effect of cooking and gastrointestinal digestion on yolk carotenoids is poorly understood. An in vitro dynamic gastrointestinal model (TIM-1) was used to investigate the digestive stability and bioaccessibility of carotenoids from boiled, fried, and scrambled eggs. Bioaccessibility but not digestive stability was significantly affected by the method of cooking. The main egg carotenoids, all-E-lutein and all-E-zeaxanthin, were stable during the digestion with average recoveries of 90 and 88%, respectively. No trans-cis isomerization of carotenoids was observed during digestion. Both all-E-lutein and all-E-zeaxanthin from scrambled eggs showed significantly lower bioaccessibility compared to boiled eggs. The results indicate that the bioaccessibility of egg carotenoids can be affected by different food preparation methods.

Determination of Differentially Expressed Genes Involved in Arabinoxylan Degradation by Bifidobacterium longum NCC2705 Using Real-Time RT-PCR

Real-time quantitative PCR (qRT-PCR) can be used to monitor specific catabolic activity by gene transcriptional analysis of bacterial cultures. This methodology has been applied to determine if the differential expression of genes putatively involved in arabinoxylan degradation by Bifidobacteriumlongum NCC2705 could be associated to the consumption of this prebiotic. Three genes putatively encoding arabinofuranosidases (abfI, abfA, and abfB) and one putatively encoding endoxylanase (xynD) were targeted for this purpose. Bifidobacteriumlongum NCC2705 exhibited higher growth yield relative to glucose based on viable counts or optical density for arabinoxylan as compared to xylose and arabinose. Among reference genes studied (16S rRNA, tufA, recA, rpoB, and atpD) the most stably expressed genes were rpoB, tufA, and atpD. The most significant increase in target gene expression was observed in the presence of arabinoxylan for the xynD gene, while xylose and arabinose had a weaker effect on xynD expression. In conclusion, B. longum NCC2705 overexpresses an endoxylanase gene in response to arabinoxylan.

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

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.

Pediococcus acidilactici UL5 and Lactococcus lactis ATCC 11454 are able to survive and express their bacteriocin genes under simulated gastrointestinal conditions

The aim of this work is to study the expression of stress genes and those involved in pediocin and nisin production in Pediococcus acidilactici UL5 and Lactococcus lactis ATCC11454 under simulated gastrointestinal (GI) physiological conditions.

Genotyping of Bifidobacterium longum subsp. longum strains by multilocus variable number of tandem repeat analysis

A multilocus variable number of tandem repeat analysis (MLVA) scheme was developed and 44 isolates of Bifidobacterium longum subsp. longum were typed, including 5 isolates recovered during a clinical trial. The MLVA scheme generated 19 profiles and proved to be a fast, reliable and relatively cheap typing method.