The immune system consists of several interdependent cells that collectively protect the body against the aggression of pathogenic microorganisms, parasites and viruses. The intestinal microflora plays an important role in the correct function of the immune system and the research carried out in this respect has shown that probiotics can modulate this aspect.

It is known that probiotics can act on the immune system of the host by signals sent from the intestinal lumen by dendritic cells, which stimulate CD4+ T cells that can lead to different populations of regulatory T cells (Treg).

Regulatory T cells are the main elements in maintaining the balance between the two populations of T helper (Th1/Th2) and, also, are responsible for maintaining the homeostasis of the immune system by secretion of IL-10.

In this regard, is important to say that all the strains present in Megaflora 9 evo have demonstrated its ability to interact with the immune system. Some of them stand out for their capacity to increase the production of IL-10, a regulatory and anti-inflammatory cytokine.

Example of mechanism regulating the host immunity by Lactobacillus plantarum W1 through interaction of dendritic cells.

Next graph shows the induction of IL-10 by different strains of Megaflora 9 evo in the presence of peripheral mononuclear blood cells. From the different tested strains, Lactobacillus plantarum W21, L. acidophilus W22 and Enterococcus faecium W54, were those that presented a higher degree of stimulation, being E. faecium W54 the strain with the greatest potential in stimulating the production of IL-10.

Production of IL-10 in a culture of peripheral mononuclear blood cells in presence of different strains present in Megaflora 9 evo (Lactococcus lactis W19, L. paracasei W20, L. plantarum W21, L. acidophilus W22 and Enterococcus faecium W54).

Maintaining intestinal homeostasis requires that the epithelium, a layer of cells lining the intestinal wall, keeps formeing an uninterrupted barrier without spaces or gaps between the enterocytes. This optimal situation is known as barrier effect.

Normally, in a healthy person there is a communication and control between the host and microbiota residing in the gut, which creates a homeostatic balance of bacterial flora by promoting the growth of beneficial bacteria, preventing overgrowth and controlling the development of pathogenic bacteria.

The phenomena of dysbiosis, or imbalance of intestinal flora, invariably lead to bacterial overgrowth of the small intestine called SIBO (Small Intestine Bacterial Overgrowth), which tilts the balance toward the growth of pathogenic bacteria.

It is also known that situations such as food intolerances, presence of allergens like gluten, digestive disorders and even lifestyle are factors that increase intestinal permeability, losing the integrity of the intestinal mucosa.

All these unfavourable conditions taken together lead to an imbalance in the intestine that causes an alteration in the absorption processes, with the consequent lack of nutrients and micronutrients in the body, essential for a healthy live and, in the more severe states, favours the passage of toxic substances into the body.

The damage caused by the intestinal imbalance not only cause situations such as irritable bowel syndrome or Crohn’s disease, but some scientific studies claim that high permeability of the intestine can be the cause of many mental and nervous illnesses, such as senile dementia, Alzheimer or Parkinson among others.

Megaflora 9 evo has demonstrated its ability to counter the phenomena of dysbiosis rebalancing the intestinal flora, maintaining the barrier effect and promoting the integrity of the gut mucosa, that allows the maintenance of the absorption processes and homeostasis between the host and the intestinal microbiota.

The integrity of the intestinal epithelium can be measured by an in vitro test that measures the Trans-Epithelial Electrical Resistance (TEER).

TEER methodology

TEER is a widely accepted quantitative technique that measures the dynamic integrity of cell junctions (tight junction) in models of endothelial and epithelial monolayers. TEER values are reliable indicators of the integrity of cell barriers for assessing the transport of drugs and other substances.

The values obtained in the TEER inform in real time reports about the integrity or alteration of monolayer cells, which is manifested by the electrical variation of values between the upper and the lower chambers. When the monolayer is subjected to the presence of a stressor in the apical part (upper chamber), the change in permeability can be measured as a decrease TEER between the upper and lower chamber.

The effectiveness of a probiotic strain on maintaining the barrier effect while placing the stressor can be detected by smaller decreases in TEER. The lesser decrease in TEER, the more effectiveness of the probiotic in maintaining the integrity of the monolayer and therefore it will preven the increase of permeability in presence of stressor agents.

TEER Methodology

To determine the barrier effect of Megaflora 9 evo, a Caco-2 monolayer cells was subjected to the presence of a stressor which increases the monolayer permeability.

The result showed that Megaflora 9 evo had a positive effect on the integrity of the monolayer cells, reducing the negative impact of the stressor compared to control.

Change in TEER in a monolayer of Caco-2 cells subjected to the presence of a stressor (red) and in the presence of the stressor and Megaflora 9 evo (green), relative to the control without stressor (blue)

Upon this result, it follows that the composition of Megaflora 9 evo has a balancing effect on the microflora in the intestine, avoiding the increased permeability of the intestinal mucosa caused by the presence of certain substances, pathogens or situations that influence the regulation of the bowel activity.

>> Action at Level 3: immunomodulation

The capacity to inhibit a pathogen, either from a bacteria or a substance with antibiotic properties, is usually determined in vitro in a growth medium with agar in Petri dish, where the pathogen is growing and where the substance or bacteria to evaluate is placed.

This classical method is based on the presence of an inhibition zone around a hole of 8 mm in which the bacteria or antibiotic substance is placed. The diameter of the inhibition zone around the hole classifies the inhibitory capacity in four grades:

• Grade 0: no inhibition zone.
• Grade 1: inhibition zone from 8 to 11 mm.
• Grade 2: inhibition zone from 11 to 14 mm.
• Grade 3: inhibition zone more than 14 mm.

The degree of inhibition is proportional to the diameter, then, the product with Grades 2 and 3 in the test are considered optimal to counteract the growth of the pathogen studied.

The following graphs show the results of inhibition of different assays with Megaflora 9 evo, or with the specific strains it contains, on various pathogens.

Megaflora 9 evo has demonstrate to have a high degree inhibiting pathogens belonging to the family Enterobacteriaceae such as E. coli, Shigella, Salmonella and Proteus agglomerans. In all cases, the degree of inhibition was 3 with a value of the inhibition zone over 14 mm.

Growth inhibition of Enterobacteriaceae

In another test, the individual inhibitory capacity of some strains of Megaflora 9 evo on the growth of Candida albicans was evaluated. The results showed different degree of inhibition depending on the strain, being L. plantarum and L. salivarius the most active. L. salivarius, in particular, showed a 100% inhibition of Candida growth. Also, Lactococcus lactis showed good inhibition with a score of more than 80%.

Growth inhibition of Candida albicans

In a third test, it was determined the inhibitory capacity of Clostridium difficile growth at 24 and 48 hours of incubation, as well as the production of toxins A and B. In this case, Enterococcus faecium W54 strain presented the most potent inhibitory capacity, with a significant inhibition of growth at 24h and total at 48h. Other strains, as L. paracasei W20 and L. salivarius W24, present in Megaflora 9 evo also inhibited the total Clostridium growth at 48 h.

Concerning the inhibition of the production of toxins A & B besides of the strains mentioned also Bifidobacrtrium lactis W51 was also able to inhibit totally the production of these enterotoxins.

It is interesting to compare the results of this study with the inhibition of Candida albicans, because it justifies the presence of multispecies and multi-strain in the mixture. While Enterococcus faecium W54 showed no inhibition on candidas but was the most active inhibiting the growth of Clostridium difficile.

Growth inhibition of Clostridium difficile

>> Action at Level 2: influence on the epithelial barrier function