Abstract
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.
Highlights
A minimum of cfu/mL or g and even higher numbers such as and cfu per daily portion of probiotic bacteria must be present in a product to retain their health benefits (Gomes da Cruz et al, 2009; Bhadoria and Mahapatra, 2011; Karimi et al, 2011)
OPTIMIZATION OF propidium monoazide (PMA)-quantitative PCR (qPCR) FOR CHEESE Cross amplification for primer and probe specificity For all primer and probe sets designed to be specific for each targeted bacterial species (Lactococcus sp., L. helveticus RO052, L. rhamnosus RO011, and B. animalis subsp. lactis BB-12), Ct values for targeted DNA were between 17 and 21, and non-targeted DNA Ct values were always above 30 with no cross amplification (Table 3)
Results obtained by PMA-qPCR were lower than those obtained by traditional microbiological methods for the first data point, indicating that culture media may lack specificity and detect nontargeted probiotic strains or non-starter lactic acid bacteria naturally found in Cheddar cheese, especially with lower numbers of viable probiotic cells at the beginning of cheesemaking
Summary
A minimum of cfu/mL or g and even higher numbers such as and cfu per daily portion of probiotic bacteria must be present in a product to retain their health benefits (Gomes da Cruz et al, 2009; Bhadoria and Mahapatra, 2011; Karimi et al, 2011). Probiotic Cheddar cheese has already been successfully manufactured for this purpose (Daigle et al, 1999; Phillips et al, 2006; Ong et al, 2007) and suitable probiotic bacteria should be carefully chosen for this type of delivery vehicle. As lactobacilli species are naturally found in this type of cheese and are resistant to acid production during cheese manufacture, some strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus plantarum have a technological advantage for use as probiotics. Lactis BB-12 is an especially acid resistant and aerotolerant strain, making it suitable for applications in such a food matrix (Roy et al, 2011). Interactions between probiotic strains, and with the lactococci Cheddar cheese starter, could affect bacterial viability in cheese samples (Gomes and Malcata, 1999; Ong et al, 2007)
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