Probio²

*CFU : colony-forming unit
**NRV : nutritional reference values

The microbiota: an organ in its own right

The digestive tract is inhabited by a colossal number of microorganisms: 10,000 to 100,000 billion bacteria, that's just a little more than the total number of cells in our body! [1]. This concentration of bacteria that cohabitates with our body is called the microbiota, which is increasingly considered an "organ" in its own right. These bacteria are mostly located in the intestine, especially in the colon (the part of the intestine commonly known as the "large intestine"), which is populated by between 1 billion and 100 billion microorganisms. But they are also found throughout the gastrointestinal tract [2], in the mouth, on the lips, on the skin, in the nose, etc.

Throughout the digestive tract, the microbiota assists our bodies in the digestion and assimilation of micronutrients. One of its best known roles is the digestion of fibre, the consumption of which is highly recommended. These fibres have polysaccharide structures (sugar chains) of varying length and are digested by our intestinal flora.

All fibres are not equal and can be categorized simply according to their solubility [3] :

• Insoluble fibres found mainly in the rigid walls of vegetables and whole grains that remain in transit without being fermented, often causing digestive discomfort.
• Soluble fibre found in fruits, vegetables and legumes is the preferred food of our microbiota and stimulates their growth. Some of them are less recommended such as the famous fructo-oligosaccharides (FOS) and inulin because they can cause bloating depending on the person.

But what happens in our colon, the main site of our microbiota, during digestion? At this level come fibers but also residual nutrients (from proteins and sugars) that our microbiota will ferment and degrade. This fermentation produces various compounds of interest, called "bacterial metabolites", which will induce local effects in our colon. Among these compounds, we particularly distinguish the "short-chain fatty acids", three in number: acetate, butyrate and propionate [4]. At the colon level, short-chain fatty acids will serve as an energy source for colon cells [5], activate the specialization of key cells (cells that produce messengers for the body) [6] and control barrier function and permeability (inflammation control, defense of foreign microorganisms, etc.) [7].

These effects translate into improved digestion, optimized metabolism, a sharper immune system, etc.

Finally, beyond the production of short-chain fatty acids through digestion, did you know that our microbiota plays an important role in the production of a variety of nutritional compounds? In fact, our intestinal bacteria participate in the production of vitamin K and certain B group vitamins such as B12 and B9 [8,9].

The microbiota: central player in our health

You've understood that a large part of our health is therefore played in our intestine. Beyond its digestive functions, our microbiota plays a key physiological role, as it has essential functions for our organism: barrier function and role in the immune system, metabolic and nervous regulation, etc. [2]. It is because of its different roles and because our microbiota is extremely adaptable (environment, diet, medication, etc.) that it is capable of playing a fundamental role in our health, and at various levels [10].

Today, scientists all agree that a "healthy", i.e. "symbiotic" microbiota contributes to the proper balance of our physiological functions beyond our digestive functions [11,12]. Conversely, the scientific literature has reported important relationships between an impoverished and disturbed microbiota (i.e. "dysbiosis") and a variety of health problems, ranging from digestive disorders to metabolic and nervous disorders [13,14].

You'll understand, it's better to take care of your microbiota. The health of the latter depends on many parameters, from diet (THE major factor), to age, through your present and past environment [15]! Indeed, more and more research is linking the child's microbiota and the mother's diet to the environment/events during the perinatal period [16,17]. Yes, you may not be wrong to ask yourself some questions about the origin of your microbiota !

Probiotics, what are they?

You often hear about probiotics, but without necessarily explaining what's behind the word. The World Health Organization (WHO) has defined probiotics as "living microorganisms that, when administered in adequate amounts, provide beneficial health effects to the host" [18]. In nutraceuticals, probiotics are essentially proposed in the form of freeze-dried bacterial strains and the number of CFU - Colony Forming Unit - is often put forward as the unit of measurement that allows the quantity of viable bacteria present in the product to be counted.

If numerous scientific data on probiotics have demonstrated their positive impact on gastrointestinal health in response to various physiological disorders (transit, irritability of the colon, etc.)[19,20], it is important to note that the number of CFU's in the product is not necessarily the same as the number of CFU's in the food supply. One of the major prerequisites of a good probiotic is its ability to persist and act throughout the intestinal tract.

As a result, the majority of nutraceutical products present seemingly balanced and highly dosed mixes (often based on Lactobacilli and Bifidobacteria) but actually hide a generic and simply profitable bacteria mix to produce, without any established scientific rationale. What is a generic bacterium? It is very simple. They are strains identified by their family name (for example Lactobacillus plantarum) without knowing exactly which family member we are talking about.

Let's say we are talking about the Dupont family. I know Mrs. Dupont from afar, and I tell myself that the Duponts probably all look alike and have the same talents. That's precisely the assumption we make when we work with generic strains. We know that members of the "Plantartum" family are generally very active in the gut, and we assume that if I take any "Plantarum" (randomly, the one available from my producer, the cheapest one), it will do the job as well as all the others. Basically, the promise of these products is: "it seems to work, but don't ask us how!"... And so much the worse for science.

Fortunately, better characterized and methodically studied products are beginning to be well documented, like our 3 star strains. In other words, we're not talking about a Mr. or Mrs. Smith, we're talking about a particular member of the family, let's say Pierre Smith... of whom we know absolutely everything: height, age, hair color... His DNA is dissected in the smallest details, and above all, we know that he's the one you need, not his brother Paul, nor his cousin Martine.

Not all probiotics are created equal. Not all probiotics are created equal. You may have noticed that in recent years, the number of probiotic formulas has increased exponentially. However, the quality of a formula remains difficult to evaluate and this is why many labels boast easy arguments, but not always true, such as the overbidding on CFU numbers, the multiplication of the number of strains or even storage in the refrigerator... All this without any clear rationale or credible explanations.

As a result, products are multiplying but the questions remain: which probiotics? What dosages for the best effectiveness? Which target population? What safety? The answers to these questions are also recommended criteria in the literature for choosing good probiotics [21].

Today, a significant gap is widening between products based on science and those based on received ideas. If in doubt, take a look at the list of strains in your mix, and ask yourself these simple questions:

• Where do the strains come from?
• Have they been studied for a specific effect?

In fact, to understand how probiotic products are generally formulated, please visit our blog: Probiotics: swiss nutraceutical knife.

Flavonoids (polyphenols), compounds with prebiotic potential ?

As if the complexity of probiotics was insufficient, we will now move on to prebiotics. And let's start with the basics: what is a prebiotic? Prebiotics have been defined as non-viable components that have the ability to reach the colon and selectively stimulate (by fermentation) the growth and/or activity of one or more colonic bacteria [22]. Simply put, this is "good food" for the bacteria in your microbiota: the better they eat, the better they do, and by immediate ricochet, the better you do !

A recent publication, however, has reported a broader definition that is beginning to gain consensus in the scientific community. It describes prebiotics as substrates ("usable" compounds) specifically used by colonic microorganisms in the host conferring a health benefit. Indeed, while it is established that prebiotics are often fibers (the best known, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin, psyllium ...), other compounds are now cited, such as polyphenols or polyunsaturated fatty acids, which may correspond to this more "modern" definition of prebiotics [23]. Why is this? Simply because they also stimulate the growth of colonic bacteria, even if they do so indirectly.

On the other hand, if compounds such as polyphenols seem to be able to interact efficiently and beneficially on the microbiota, their structure must be considered. For although they show promising activity in test tubes, however, they often fail in their ability to act "in real life" or in vivo. It should be noted that polyphenols are simply completely metabolized (the molecule is broken down by the body and evacuated) before they reach their place of action. These molecules are therefore very beneficial at the level of the colon [24], provided that they can get there...

A specific selection of certain polyphenols is necessary to ensure that they reach the colon [25]. Fortunately, nature does things well, since some polyphenols are naturally protected (dare we say vectorized!) and can reach the colon intact to be metabolized there and act. They are mainly found in citrus fruits (oranges, grapefruit, etc.): the famous flavonoids (hesperidin, for example).

Putting science back at the heart of product development

If a gap is growing between standard formulas and those that are in line with the latest scientific advances, then we clearly choose to be in the second category. Probio² puts science back at the centre of its design, by adhering to the latest advances in science, down to the smallest detail.

The combination of our 5 lactobacilli and 4 bifidobacteria has been designed to bring together the best of both philosophies:

• Specific and targeted actions with 3 star strains with multiple studies. Judge for yourself: 21 clinical studies leading to the filing of 22 patents. Strains identified since the early 1990s, extensively studied and characterized.
• 6 complementary strains for diversity. While targeted effects are gaining momentum, many studies continue to show the benefits of balanced mixes [26,27].

Close-up on our 3 star strains

Lactobacillus plantarum 6595 (Probi : DSM 6595)

- Identity : dominant species within the genus Lactobacillus that colonize our digestive tract [28]
- Origin of strain: human intestinal mucosa
- Science: 9 clinical studies on a total of 743 subjects
- Patents: 11 patents to its credit
- Setback: subject of study since 1993 and safety status recognized by European authorities (QSP status)
- Strength: intestinal and digestive health

The beneficial effects of Lactobacillus plantarum 6595 on intestinal health result from its significant capacity to adhere to the cells of the intestinal tract [29,30]. In particular, this strain has shown effects on intestinal protection in weakened subjects: the famous barrier function of the intestine [31-34].

Lactobacillus rhamnosus 6594 (Probi : DSM 6594)

- Identity : species colonizing our digestive tract that can be associated with breastfeeding during childhood [35]
- Origin of strain: human intestinal mucosa
- Science: 4 clinical studies on a total of 769 subjects
- Patents: 4 patents to its credit
- Setback: subject of study since 1993 and safety status recognized by the European authorities (QSP status)
- Strength: intestinal barrier function

Lactobacillus rhamnosus 6594 has an intestinal adhesion capacity [29,36], and draws its strengths from its ability to act at the level of permeability and inflammatory response at the intestinal level. Lactobacillus rhamnosus is also the strain found in the majority of intestines of healthy newborns. It remains in the majority in the intestine during childhood, and is useful throughout life [36,37].

Lactobacillus paracasei 13434 (Probi : DSM 13434)

- Identity : species of the Lactobacillus group mainly used in the food industry for the elaboration of our daily products [38]
- Origin of the strain: human intestinal mucosa
- Science: 8 clinical studies on a total of more than 860 subjects
- Patents: 7 patents to its credit,
- Setback: subject of study since 1998 and safety status recognized by the European authorities (QSP status),
- Strength: immune response

Shown to have a survival capacity in the intestinal tract [39,30], Lactobacillus paracasei 13434 is involved in the modulation of the immune response and in the efficacy of the response. In a clinical study, it showed an increase in immune defence activity (production of immune molecules) in subjects who received it orally for two weeks [40].

These strains show actions on intestinal health and disorders related to its deregulation. This combination offers an approach on the 3 axes of local functioning of the intestinal tract: barrier, immune and digestive functions.

A polyphenol with proven prebiotic potential

Polyphenols have been reported in the scientific literature as "functional food components that can influence our microbiota" [41]. But although they are able to interact with our microbiota in a beneficial way, their arrival in the colon is not always proven. We have therefore naturally turned to citrus flavonoids, which are naturally armed to survive and reach the colon intact.

Microbiomex® is a citrus extract (oranges and grapefruit) rich in flavonoids that has proven its action on the activity of the intestinal microbiota in in vivo and clinical studies, i.e. in real life. The glycosylated chemical form (associated with sugars) of the flavonoids contained in this extract, allows them to be protected in the digestive tract to be finally degraded (cleavage of the bond with sugars = deglycosylation) at the colonic level.

The results of the studies carried out on Microbiomex® have shown that it potentiates the production of short chain fatty acids suggesting a modulation of the activity of the microbiota as early as 500mg [42]. This 100% vegetable extract, standardised in hesperidin, is thus attributed a prebiotic action according to the most recent definitions.

Flavonoids act by two mechanisms:

• as a substrate for the microbiota (prebiotic action),
• an effect on inflammation [43].

A meaningful Probio/Prébio combination

As you will have understood, the decisive advantage here is that the beneficial effect on the microbiota (prebiotic) is obtained at a very low dosage compared to that of fibres (7g), prebiotics of another category. As a result, the Probiotic/Prebiotic mix fits in 2 small daily capsules (and not just any capsules, we will see later).

And as you follow perfectly, you logically wonder if the prebiotic does not represent a risk for the stability of the probiotics, since they are mixed in the same capsule. Well, we asked ourselves the same question, and the answer is "no", no stability risk for probiotics, we prove it to you by studies (science, always science).

Guaranteed effectiveness over time

First Guarantee: A "controlled" dosage

On the market, many probiotic formulas confuse the message and promote a probiotic mix whose dosages are not very explicit. Very often, the diversity of strains takes precedence over their stability and resistance. As a result, you have the right to ask yourself what guarantees can be used to ensure that you will benefit from the promised effects ?

A probiotic mix can only be used for a dosage that combines a sufficient quantity of CFU (dose-response) and viability (dose-stability). The star strains we have selected have shown very good stability and survival through the gastrointestinal tract.

We have also multiplied by 12.5 the recommended dosage of our 3 star strains to ensure the recommended dose at the expiry date of the product. Indeed, it is very complex to follow the viability of the strains throughout the life of the product, which is why a dose of 12.5 billion per strain ensures that Probio² provides at least the recommended dose.

*10⁹ : 1 billion
** CFU : Colony Forming Unit

Second guarantee: Encapsulation in DRcaps®*

DRcaps® (Capsugel) is a Made In France technology of HMPC (vegetable hypromellosis, fibers) capsules developed to effectively protect compounds sensitive to gastric acidity. Its effectiveness in terms of the release of compounds after passing through the stomach (acid medium) has been demonstrated by a simulation study of the human digestive tract [44] and also in clinical studies. These data showed that the complete dissolution of the capsule, and thus the release of the nutritional compounds present in the capsule, took place in the intestine (small intestine, just after the stomach).
*DRCaps® is a trademark of LONZA (or one of its subsidiaries), registered in the EU.

Third guarantee: production by world specialists

Our desire to produce the best probiotics on the market has inevitably led us to seek out the best specialists at international level.

- Probi supplies us with probiotics. He is a world-renowned expert since the 1980s. The strains he produces himself have been studied since the 1990s and enjoy an unparalleled degree of hindsight, both in terms of effectiveness and stability.
- Our processor, who encapsulates these probiotics, also enjoys a worldwide reputation in the field. Nutrilinea perfectly controls the production of probiotic capsules, in particular by applying an exceptional level of control of Temperature and Humidity conditions :20/20 = "20°/20%". World record !

Publications

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3. Dai, F.-J.; Chau, C.-F. Classification and regulatory perspectives of dietary fiber. J. Food Drug Anal. 2017, 25, 37–42.
4. Rosenbaum, M.; Knight, R.; Leibel, R.L. The gut microbiota in human energy homeostasis and obesity. Trends Endocrinol. Metab. 2015, 26, 493–501.
5. Tremaroli, V.; Bäckhed, F. Functional interactions between the gut microbiota and host metabolism. Nature 2012, 489, 242–249.
6. Bauer, P.V.; Hamr, S.C.; Duca, F.A. Regulation of energy balance by a gut–brain axis and involvement of the gut microbiota. Cell. Mol. Life Sci. 2016, 73, 737–755.
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8. Rossi, M.; Amaretti, A.; Raimondi, S. Folate Production by Probiotic Bacteria. Nutrients 2011, 3, 118–134.
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11. Sekirov, I.; Russell, S.L.; Antunes, L.C.M.; Finlay, B.B. Gut Microbiota in Health and Disease. Physiol Rev 2010, 90, 46.
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14. Cani, P.D. The gut microbiota manages host metabolism. Nat. Rev. Endocrinol. 2014, 10, 74–76.
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17. Sprockett, D.; Fukami, T.; Relman, D.A. Role of priority effects in the early-life assembly of the gut microbiota. Nat. Rev. Gastroenterol. Hepatol. 2018, 15, 197–205.
18. Probiotics in food: health and nutritional properties and guidelines for evaluation; FAO, World Health Organization, Eds.; FAO food and nutrition paper; Food and Agriculture Organization of the United Nations : World Health Organization: Rome, 2006; ISBN 978-92-5-105513-7.
19. Wilkins, T. Probiotics for Gastrointestinal Conditions: A Summary of the Evidence. 2017, 96, 10.
20. Kechagia, M.; Basoulis, D.; Konstantopopoulou, S.; Dimitriadi, D.; Gyftopoulou, K.; Skarmoutsou, N.; Fakiri, E.M. Health Benefits of Probiotics : A Review. ISRN Nutr. 2012, 2013, 7.
21. Sarao, L.K.; Arora, M. Probiotics, prebiotics, and microencapsulation: A review. Crit. Rev. Food Sci. Nutr. 2017, 57, 344–371.
22. Ziemer, C.J.; Gibson, G.R. An Overview of Probiotics, Prebiotics and Synbiotics in the Functional Food Concept: Perspectives and Future Strategies. Int. Dairy J. 1998, 8, 473–479.
23. Gibson, G.R.; Hutkins, R.; Sanders, M.E.; Prescott, S.L.; Reimer, R.A.; Salminen, S.J.; Scott, K.; Stanton, C.; Swanson, K.S.; Cani, P.D.; et al. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nat. Rev. Gastroenterol. Hepatol. 2017, 14, 491–502.
24. Oteiza, P.I.; Fraga, C.G.; Mills, D.A.; Taft, D.H. Flavonoids and the gastrointestinal tract: Local and systemic effects. Mol. Aspects Med. 2018, 61, 41–49.
25. Cardona, F.; Andrés-Lacueva, C.; Tulipani, S.; Tinahones, F.J.; Queipo-Ortuño, M.I. Benefits of polyphenols on gut microbiota and implications in human health. J. Nutr. Biochem. 2013, 24, 1415–1422.
26. Chapman, C.M.C.; Gibson, G.R.; Rowland, I. Health benefits of probiotics: are mixtures more effective than single strains? Eur. J. Nutr. 2011, 50, 1–17.
27. Ouwehand, A.C.; Invernici, M.M.; Furlaneto, F.A.C.; Messora, M.R. Effectiveness of Multi-strain Versus Single-strain Probiotics: Current Status and Recommendations for the Future. J. Clin. Gastroenterol. 2018, 52, S35–S40.
28. Vries, M.C. de Analyzing global gene expression of Lactobacillus plantarum in the human gastrointestinal tract; Wageningen, 2006; ISBN 978-90-8504-344-7.
29. Johansson, M.L.; Molin, G.; Jeppsson, B.; Nobaek, S.; Ahrné, S.; Bengmark, S. Administration of different Lactobacillus strains in fermented oatmeal soup: in vivo colonization of human intestinal mucosa and effect on the indigenous flora. Appl. Environ. Microbiol. 1993, 59, 15–20.
30. Berggren, A.; Lazou Ahrén, I.; Larsson, N.; Önning, G. Randomised, double-blind and placebo-controlled study using new probiotic lactobacilli for strengthening the body immune defence against viral infections. Eur. J. Nutr. 2011, 50, 203–210.
31. Rayes, N.; Hansen, S.; Seehofer, D.; Müller, A.R.; Serke, S.; Bengmark, S.; Neuhaus, P. Early enteral supply of fiber and Lactobacilli versus conventional nutrition: a controlled trial in patients with major abdominal surgery. Nutrition 2002, 18, 609–615.
32. Rayes, N.; Seehofer, D.; Hansen, S.; Boucsein, K.; Müller, A.R.; Serke, S.; Bengmark, S.; Neuhaus, P. Early enteral supply of lactobacillus and fiber versus selective bowel decontamination: a controlled trial in liver transplant recipients: Transplantation 2002, 74, 123–128.
33. Oláh, A.; Belágyi, T.; Issekutz, A.; Gamal, M.E.; Bengmark, S. Randomized clinical trial of speci®c lactobacillus and ®bre supplement to early enteral nutrition in patients with acute pancreatitis. Br. J. Surg. 2002, 5.
34. Klarin, B.; Molin, G.; Jeppsson, B.; Larsson, A. Use of the probiotic Lactobacillus plantarum 299 to reduce pathogenic bacteria in the oropharynx of intubated patients: a randomised controlled open pilot study. Crit. Care 2008, 12, R136.
35. Kant, R.; Rintahaka, J.; Yu, X.; Sigvart-Mattila, P.; Paulin, L.; Mecklin, J.-P.; Saarela, M.; Palva, A.; von Ossowski, I. A Comparative Pan-Genome Perspective of Niche-Adaptable Cell-Surface Protein Phenotypes in Lactobacillus rhamnosus. PLoS ONE 2014, 9, e102762.
36. Lazou Ahrén, I.; Berggren, A.; Teixeira, C.; Martinsson Niskanen, T.; Larsson, N. Evaluation of the efficacy of Lactobacillus plantarum HEAL9 and Lactobacillus paracasei 8700:2 on aspects of common cold infections in children attending day care: a randomised, double-blind, placebo-controlled clinical study. Eur. J. Nutr. 2020, 59, 409–417.
37. Pärtty, A.; Kalliomäki, M.; Endo, A.; Salminen, S.; Isolauri, E. Compositional Development of Bifidobacterium and Lactobacillus Microbiota Is Linked with Crying and Fussing in Early Infancy. PLoS ONE 2012, 7, e32495.
38. Huys, G.; D’Haene, K.; Danielsen, M.; Mättö, J.; Egervärn, M.; Vandamme, P. Phenotypic and Molecular Assessment of Antimicrobial Resistance in Lactobacillus paracasei Strains of Food Origin. J. Food Prot. 2008, 71, 339–344.
39. Antonsson, M. Lactobacillus strains isolated from Danbo cheese as adjunct cultures in a cheese model system. Int. J. Food Microbiol. 2003, 85, 159–169.
40. Rask, C.; Adlerberth, I.; Berggren, A.; Ahrén, I.L.; Wold, A.E. Differential effect on cell-mediated immunity in human volunteers after intake of different lactobacilli: Lactobacilli and cell-mediated immunity. Clin. Exp. Immunol. 2013, 172, 321–332.
41. Laparra, J.M.; Sanz, Y. Interactions of gut microbiota with functional food components and nutraceuticals. Pharmacol. Res. 2010, 61, 219–225.
42. Salden, B.N.; Troost, F.J.; Possemiers, S.; Stevens, Y.; Masclee, A. Maastricht Univ. Maastricht NetherlandsUnpublished Work 2019.
43. Stevens, Y.; Rymenant, E.V.; Grootaert, C.; Camp, J.V.; Possemiers, S.; Masclee, A.; Jonkers, D. The Intestinal Fate of Citrus Flavanones and Their Effects on Gastrointestinal Health. Nutrients 2019, 11, 1464.
44. Marzorati, M.; Possemiers, S.; Verhelst, A.; Cadé, D.; Madit, N.; Van de Wiele, T. A novel hypromellose capsule, with acid resistance properties, permits the targeted delivery of acid-sensitive products to the intestine. LWT - Food Sci. Technol. 2015, 60, 544–551.